Next Article in Journal
The Role of Natural Compounds in Optimizing Contemporary Dental Treatment—Current Status and Future Trends
Next Article in Special Issue
XPS Characterization of TiO2 Nanotubes Growth on the Surface of the Ti15Zr15Mo Alloy for Biomedical Applications
Previous Article in Journal
Fabrication of Biomedical Ti-Zr-Nb by Reducing Metal Oxides with Calcium Hydride
 
 
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Dentin, Dentin Graft, and Bone Graft: Microscopic and Spectroscopic Analysis

by
Elio Minetti
1,†,
Andrea Palermo
2,†,
Giuseppina Malcangi
3,
Alessio Danilo Inchingolo
3,
Antonio Mancini
3,
Gianna Dipalma
3,*,
Francesco Inchingolo
3,*,
Assunta Patano
3,*,‡ and
Angelo Michele Inchingolo
3,‡
1
Department of Biomedical, Surgical, Dental Science, University of Milan, 20161 Milan, Italy
2
College of Medicine and Dentistry, Birmingham B4 6BN, UK
3
Department of Interdisciplinary Medicine, University of Bari “Aldo Moro”, 70124 Bari, Italy
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work as first authors.
These authors contributed equally to this work as last authors.
J. Funct. Biomater. 2023, 14(5), 272; https://doi.org/10.3390/jfb14050272
Submission received: 29 March 2023 / Revised: 1 May 2023 / Accepted: 12 May 2023 / Published: 13 May 2023

Abstract

:
Background: The use of the human dentin matrix could serve as an alternative to autologous, allogenic, and xenogeneic bone grafts. Since 1967, when the osteoinductive characteristics of autogenous demineralized dentin matrix were revealed, autologous tooth grafts have been advocated. The tooth is very similar to the bone and contains many growth factors. The purpose of the present study is to evaluate the similarities and differences between the three samples (dentin, demineralized dentin, and alveolar cortical bone) with the aim of demonstrating that the demineralized dentin can be considered in regenerative surgery as an alternative to the autologous bone. Methods: This in vitro study analyzed the biochemical characterizations of 11 dentin granules (Group A), 11 demineralized using the Tooth Transformer (Group B), and dentin granules and 11 cortical bone granules (Group C) using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS) to evaluate mineral content. Atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared by the statistical t-test. Results: The significant p-value (p < 0.05) between group A and group C indicated that these two groups were not significantly similar, while the non-significant result (p > 0.05) obtained between group B and group C indicated that these two groups are similar. Conclusions: The findings support that the hypothesis that the demineralization process can lead to the dentin being remarkably similar to the natural bone in terms of their surface chemical composition. The demineralized dentin can therefore be considered an alternative to the autologous bone in regenerative surgery.

1. Introduction

The tooth grafting process has been introduced by Urist et al. more than 50 years ago, when they uncovered the osteoinductive properties of the demineralized dentin matrix [1,2]. Fundamental growth agents for bone regeneration can be found in both the bone and the dentin matrix. These biological materials have been suggested as potential graft materials by several authors. Bone is composed of 61% inorganic mineral, and 39% organic substance and water [3,4,5,6].
The organic part (39%) is made up of 90% collagen type I, with the remaining 10% being made up of non-collagen proteins produced by the bone cells. Ten percent of the total organic content is then incorporated into the bone matrix during the process of osteosynthesis [7].
The Ca/P molar ratio of bone apatite nanocrystals differs from the stoichiometric hydroxyapatite ratio of 1.67 due to a variety of substitutions and vacancies. The crystals in the bone (low crystalline calcium phosphate) are nanosized, measuring 20–60 nm in length and 5–20 nm in breadth [8,9,10].
Some of the important non-collagenous proteins are osteocalcin (Gla proteins), osteopontin, bone sialoproteins, and osteonectin. The bone matrix also contains proteoglycans, bone morphogenetic proteins (BMPs), and a variety of growth factors, including platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), and insulin-like growth factors (IGF). Moreover, lysyl oxidase and tyrosine-rich acidic matrix proteins (TRAMP) are further important components of the demineralized bone matrix. Growth factors are fixed and stored in enormous concentrations in the mineralized matrix of bone. According to this hypothesis, growth factors such as IGF-II are deposited for a period of time before being released in a bioactive form by osteoblast bone resorption to act on pre-osteoblasts and mature osteoblasts, thereby allowing for the site-specific replacement of bone lost to resorption [2,11,12,13].
Dentin and cement are two different specialized forms of bone. The dentin is harder than the compact bone, and is made up of organic matrix (28%) and inorganic matrix (72%) [14], and these crystals are the same size for dentin and bone [15,16].
Dentin consists of 35% organic matter (90% collagen type 1, with the remaining 10% non-collagenous proteins including BMPs), water, and 65% inorganic material (hydroxyapatite in high crystalline calcium phosphate). The majority of dentin is made up of proteins that are found in both the dentin and bone [17,18].
Proteins common in both bone and dentin include: collagen types I, III, and V, bone sialoprotein (BSP), osteopontin (OPN), dentin matrix protein-1 (DMP-1), osteocalcin (OC), and osteonectin (ON) [17,19].
The organic substance (making up 10% of the 35% organic matter) comprises collagenous fibrils embedded in mucopolysaccharide ground substance. The main kind of collagen found in dentin is type I collagen. The matrix is synthesized by odontoblasts and is a rich source of growth factors and contains bioactive molecules required for dentinogenesis. These molecules are released in the presence of bacterial acids or certain dental materials in the case of caries or restorative treatments, causing dentin regeneration and repair [17,20].
Proteoglycans such as chondroitin sulfates, decorin, and biglycan; glycoproteins including dentin sialoprotein (DSP), osteonectin, and osteopontin; phosphoproteins such as dentin phosphoprotein (DPP), and gamma carboxy-glutamate containing proteins (GLA-proteins), and phospholipids, are important constituents of the ground substance. Dentin matrix and bone protein are similar; however, dentin sialoprotein and dentin phosphoprotein are uniquely found in dentin. TGF, FGF, IGFs, BMPs, epidermal growth factor (EGF), PDGF, placenta growth factor (PLGF), vascular endothelial growth factor (VEGF), and angiogenic growth factor (AGF) are all found in the matrix. These matrix components play critical roles in dentin mineralization, and include various growth factors including transforming growth factor-b (TGF-b1), IGF, BMPs, and several angiogenic growth factors [21,22,23,24].
Dentin represents an efficient source of BMPs, bioactive growth factors (GFs), and transforming growth factor-B (TGF-B), which all play a role in bone repair processes [25].
The first to theorize the presence of GFs in dentin was Urist in 1971, who stated that the BMPs, which stimulate bone formation, are found in dentin, as are non-collagenous proteins such as osteocalcin, osteonectin, and dentin phosphoprotein [26].
According to experts, the demineralization process provides superior bone augmentation compared to non-demineralized dentin [27,28].
A tooth graft without any treatment is contaminated and is not safe to use it in surgical procedure [29], but recently an innovative medical device was introduced to the market that is able to automatically clean and demineralize, as well as obtain suitable tooth graft materials starting from the whole tooth of the patient. In vitro investigations on the graft material generated by this innovative technology showed that the demineralization process increases BMP-2 bioavailability [30,31].
BMP-2 is important in the control of odontoblast differentiation and dentin production [32,33,34,35]. Nampo et al. in 2010 analyzed all the growth factors presence in both the dentin and bone [36].
BMP-2 significantly increases bone growth in the demineralized dentin matrix (DDM) carrier system [4,19,30,37].
The mineral component is composed of hydroxyapatite crystals, with carbonate content and a lower Ca/P ratio than the pure hydroxyapatite. The inorganic component consists of hydroxyapatite, as in bone. The crystals are plate-shaped and much smaller than the hydroxyapatite crystals in enamel but are ten times bigger than the bone. The incremental lines of von Ebner reflect the daily rhythmic, recurrent deposition of the dentin matrix. The course of the lines indicates the growth pattern of the dentin. The daily deposition is approximately 4 µm. Dentin is similarly mineralized in a 12 h cycle [38,39,40,41].
Crystals of hydroxyapatite (HA) are present in the bone and in the dentin in the shape of plates or needles. These crystals are round long, wide, and thick (Table 1). The major mineral component of teeth and bones is hydroxyapatite (Ca10(PO4)62(OH). The structure of bone contains around 65 wt.% hydroxyapatite, a needle-shaped compound with a length of 20–60 nm and a width of 5–20 nm that is responsible for stiffness and strength. Using X-ray diffraction (XRD), the average size of hydroxyapatite crystallites in dentin was estimated. The crystallites were found to be in the form of flattened plates, 80 + −12 nm in length, 3–4 nm in thickness, and 40 + −10 nm in width [42,43].
The aim of this study was to compare the mineral content, using scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), of the dentin, the demineralized dentin, and the cortical bone. We aimed to analyze these three samples to evaluate both the similarities and differences with a particular interest on demineralized dentine vs. bone to understand whether the demineralization treatment makes the demineralized dentin similar to the bone.

2. Materials and Methods

Twenty-two dentin samples and eleven bone samples were collected from patients according to the Declaration of Helsinki guidelines and was approved by the local Ethical Committee for Biomedical Research of Chieti and Pescara (Prot. N. 1869/21.03.2019). The bone samples, with signed patient informed consent, were taken from broken bone residues remaining attached to the extracted teeth roots or collected at the time of implants insertion. The study included patients over 18 years of age who needed a tooth extraction treatment, in good health condition (ASA-1 and ASA-2) without any alteration of bone metabolism condition and without any pharmacological use, and who were able to undergo dental surgical and restorative procedures. Tooth extractions were needed for trauma, caries, or periodontal diseases.
The dentin samples were randomly subdivided into two groups. group A (dentin without demineralization), and group B (dentin with demineralization). Following the Minetti’s group indications, teeth with root canal treatments were also utilized as in these studies it was shown that there were no significant differences in results [3].
The demineralization process was made using the innovative medical device Tooth Transformer® (TT, from Tooth Transformer® Srl, via Washington 59 20146, Milan, Italy), which can collect acceptable tooth graft materials beginning with the entire tooth. This technology ensures thoroughly automated cleaning, grinding, and demineralizing procedures, with no errors caused by human intervention. The Tooth Transformer is a cutting-edge tissue engineering device, as it can quickly process and turn a removed tooth into clinically viable bone graft material [44].
A piezoelectric tool (Mectron, Carasco (GE), Italy) was used to clean the entire excised tooth of remaining calculus. The root surface was polished with a diamond drill (ref.6855 Dentsply Maillefer, Ballaigues, Switzerland), and the tooth’s filling materials (guttapercha, composite, etc.) were carefully removed. The tooth was then chopped into little pieces and placed into the device’s mill. The single-use unit was opened, and a little box containing disposable liquids was inserted at the correct position in the device (indicated by arrows). The device was started using the general switch button once all the components were installed and the machine’s cover was closed, and the demineralized dentin graft was ready. The bone was the third group (group C).
The bone samples (eleven samples from the lower molar) were collected at the time of the implant’s insertion. After a signed consensus from the patient, a 3 mm trephine bur (MEISINGER USA, L.L.C. 10150 E. Easter Avenue Centennial, CO 80112, USA) was used to prepare the implant site, and then the dedicated implants drills were used under extensive irrigation with saline solution. The bone removed to create a surgical implant alveolus was collected. The sample was then washed with physiological solution to carefully remove any blood residues or other tissue frustules and was immediately inserted into the freshly prepared fresh fixative solution and stored away from light (10% neutral buffered formalin) in a flask of at least 10 cc of hermetic sealant volume without bubbles.
Each sample of each group was analyzed using a scanning electron microscopy (SEM) device.
An environmental scanning electron microscope (ESEM Zeiss EVO50, Carl Zeiss, Milan, Italy) linked to a secondary electron detector for energy dispersive X-ray (Carl Zeiss, Milan, Italy) nalysis was used to analyze the surface morphology of Group A-B-C sample particles.
Following a 2 h fixation with 1.5% (v/v) glutaraldehyde fixation and dehydration in progressive ethanol, samples were gold-sputtered, mounted on scanning electron microscopy (SEM) stubs, and then evaluated with a 15 kV acceleration voltage. SEM pictures were captured at a magnification of 5000. All the particles from each group were examined with EDS to analyze the surface composition (atomic percentages of carbon, oxygen, phosphorus, and calcium).

3. Results

A total of 33 analysis were performed: 22 dentin samples were analyzed from 22 different extracted teeth, 11 bone biopsies of different sites from 11 subjects (6 male and 5 women) aged 53.8 + −6.56 were performed.
The sample surfaces were found to be different between the various groups as shown in the pictures (Figure 1, Figure 2 and Figure 3).
The summary of the results of the EDS analysis are presented in Table 2 and Table 3. Statistical analysis was conducted between group A (non-treated dentin) vs. group C (bone), and between group B (demineralized dentin) and group C (bone). No statistical differences between group B (demineralized dentin) and group C (bone) were found. There was, however, a statistical difference observed between Group A (non-treated dentin) and group C (bone).

4. Discussion

Vertebrates originate from three germ layers: the ectoderm, mesoderm, and endoderm, as well as neural crest cells, which arise from the neural tube fusion region, and both the teeth and the alveolar bone are formed by neural crest cells and contain type I collagen. The maxillofacial bones (except for the occipital, sphenoid, temporal, and ethmoid bones), cartilage, teeth, and nerve and glial cells are all produced from the neural crest [36,45].
There are a lot of similarities displayed between the information presented in Table 4 and Table 5. At the tissue scale, the bone and dentin tissues are different, but at the ultrastructural scale both are made with the same constituents.
The primary morphologic distinction between the bone and the dentin is that certain osteoblasts occur on the surface of bone, and when one of these cells becomes encased within its matrix, it is then referred to as an osteocyte. Meanwhile, the cell bodies of odontoblasts remain external to dentin [46]. Dentin has the same chemical composition as bone and is regarded a suitable bone substitute. The mineralization of both bone and dentin is conducted in the same way [47,48,49].
The mineralization process includes proteins deposited into both matrices, and the impact of this process is the presence of the same proteins embedded.
In general, biomineralization consists of the process by which cells organize mineral deposition. It represents the cell-mediated process in which HA is deposited into the extracellular matrix (ECM) of the skeletal structures present in vertebrates. ECM structural molecules, along with several enzymes, direct mineral salt entrance and fixing exclusively in the bone and mineralized tooth tissues [49].
Table 6 describes the proteins that can be found in the bone and dentin.
The bone graft was used because it is the same tissue. Socket grafting has been demonstrated to be more effective in terms of bone quantity and quality than excision alone [50].
The autologous bone graft contains osteogenic properties (marrow-derived osteoblastic cells as well as preosteoblastic precursor cells), osteoinductive properties (non-collagenous bone matrix proteins, including growth factors), and osteoconductive properties (bone mineral and collagen). Being an autologous graft, there is no risk of disease transmission and perfect histocompatibility. However, there are some disadvantages to using autogenous bone, such as insufficient graft material, the possibility of significant postsurgical morbidity at the donor site (e.g., rib, fibula, or iliac crest), such as infection, pain, hemorrhage, muscle weakness, and nerve injury, increased surgical time and blood loss, and additional cost (Table 6) [51].
While deciding where to collect bones, the amount and quality of donor bone sites should also be evaluated (Table 7) [52].
There are certain disadvantages to using autogenous bone, such as insufficient graft material, due to the need for an additional surgical site. Therefore, the possibility to utilize different materials was evaluated. In particular, the dentin, as shown before, has the same properties of the bone and was previously considered as an efficient graft. Using an autologous extracted tooth there is a complete histocompatibility and no opportunity for disease transmission.
The guided bone regeneration (GBR) healing phases are similar between the dentin and the bone because they are remarkably similar autologous tissues.
Phase 1
4–6 WEEKS
During the first week after surgery, the graft is surrounded by a slew of inflammatory cells such as lymphocytes, plasma cells, osteoclasts, mononuclear cells, and polynuclear cells. There is also some fibrous tissue present. In the second week, fibrous granulation tissue predominates at the recipient location, and osteoclastic activity increases. Invading macrophages destroy the necrotic tissue within the graft’s haversian canals, resulting in the production of intracellular byproducts that, combined with the recipient site’s low oxygen tension and pH, function as a chemoattractant to host undifferentiated stem cells. Mesenchymal cells begin to develop into osteogenic cells when exposed to osteoinductive stimuli. The first phase of bleeding, inflammation, revascularization, and osteoinduction proceeds as a continuous process, with active bone production and resorption occurring within four weeks of implantation. Cancellous autografts are then integrated into a necrotic bed by the creation of new bone. As a result, the construct’s mechanical qualities are initially strengthened. The mechanical strength of the graft–host interface eventually returns as necrotic bone is resorbed and replaced [10,14].
4–6 WEEKS
Formation of the clot and migration of the vascular structures in the bone walls around the defect. The deposition of the osteoid bone starts. The cellular and molecular cascades involve cell migration from the surrounding tissue. The cells secrete factors that are essential for bone formation and remodeling. This favors the mature remodeled bone development in the underlying defect by activating the activity of osteoblasts and osteoclasts [14].
Phase 2
8–12 WEEKS
Maturation of the osteoid bone occurs and cortical bone development begins. The marrow bone will be mineralized from the osteoblasts. New cortical bone will begin to form on its periphery [14].
Phase 3
12–16 WEEKS
Maturation of the cortical bone occurs, and the remodeling of the marrow and cortical bones begin. Viewing near the membrane can permit observations of the newly modeled cortical bone [53,54].
Recently, there has been a lot of interest in autologous bone-like materials as potential substrates for bone regeneration of alveolar lesions. More particularly, the utilization of tooth-derived materials has recently piqued the curiosity of many individuals due to the natural abundance of teeth that are pulled every day and abandoned as trash [3,27].
In histology books, the dentin and the tooth cementum are two specialized forms of bone tissue. Dentin is a special form of bone tissue that is harder than compact bone [14].
Physically and chemically, dentin resembles bone. The final components of the tooth and bone are the same: collagen, hydroxyapatite, and non-collagenic proteins. In fact, dentin, in the form of native dentin and dentin derivatives, such as demineralized and deproteinized dentin have thus been used as graft materials in bone repair processes. Some experimental evidence issued from a critical literature review highlights the role of demineralized dentin matrix in stimulating osteodifferentiation in vitro and, in increasing in vivo osteoinduction [46,55,56,57,58].
The presence of growth factors, and the building of both the bone and tooth with collagen type 1, and the same inorganic molecule of hydroxyapatite Ca5(PO4)3(OH), suggests to presume possible, after following the correct procedure about disinfection and cleaning to use the tooth as a bone graft [22,59,60].
The treatment submitted from the tooth will help determine the minor or major presence of growth factors. In fact, studies from Bono and Candiani reveal the different GFs present depending on the different liquids [30].
If the dentin is considered similar to the bone, the demineralized dentin should therefore be considered very similar to the bone. The limitation of this in vitro test is the limited numerosity, and it would be far more optimal to obtain these results in vivo. These in vitro tests conclude that there are no statistical differences present between group B and Group C, and that the ratio between Ca/P is remarkably similar.

5. Conclusions

The treated dentin components values are remarkably similar to the bone component values. Tests were conducted to demonstrate that the activity carried out by the device (automated treatment with HCL and H2O2, along with a different temperature and UVA) allows a modification of the chemical compositions of the dentin to make it statistically similar to bone tissue more than the dentin without treatment. This clearly implies that the tooth can be used by means of a dedicated medical device that guaranteed the treatment in an automatic, repeatable way, and as an alternative to the use of the autologous bone in regenerative treatment.
The autologous ooth has already been used to make sinus lifts, GBR, and alveolar ridge preservation socket preservation.
More studies and different numerosities are necessary to better understand the influence of the treatment in tooth grafting.

Author Contributions

Conceptualization, E.M., A.P. (Andrea Palermo), A.P. (Assunta Patano) and A.M.I.; Methodology, E.M., A.P. (Andrea Palermo) and F.I.; Software, A.D.I. and A.M.I.; Validation, A.P. (Assunta Patano) and A.M.I.; Formal analysis, A.P. (Assunta Patano) A.M. and F.I.; Resources, A.M., G.M. and G.D.; Data curation, G.M., A.D.I. and F.I.; Writing—original draft preparation, E.M. and A.P. (Andrea Palermo); Writing—review and editing, A.P. (Assunta Patano), and A.M.I.; Supervision, G.M., A.P. (Assunta Patano), A.D.I. and G.D.; Project administration, E.M., A.P. (Andrea Palermo), F.I. and G.D. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

This research was approved by the local Ethical Committee for Biomedical Research of Chieti and Pescara (Prot. N. 1869/21.03.2019).

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Abbreviations

PDGFPlatelet-derived growth factor
FGFFibroblast growth factor
IGFInsulin-like growth factor
TRAMPTyrosine-rich acidic matrix protein
BMPBone morphogenetic protein
BSPBone sialoprotein
OPNOsteopontin
DMP-1Dentin matrix protein-1
OCOsteocalcin
ONOsteonectin
ELISAEnzyme-linked immunosorbent assay
TGFTransforming growth factor
DSPDentin sialoprotein
DPPDentin phosphoprotein
GLA-proteinsGamma-carboxyglutamate-containing proteins
EGFEndothelial growth factor
PLGFPlacenta growth factor
VEGFVascular endothelial growth factor
AGFAngiogenic growth factor
GFGrowth factor

References

  1. Yeomans, J.D.; Urist, M.R. Bone Induction by Decalcified Dentine Implanted into Oral, Osseous and Muscle Tissues. Arch. Oral Biol. 1967, 12, 999–1008. [Google Scholar] [CrossRef] [PubMed]
  2. Bang, G.; Urist, M.R. Bone Induction in Excavation Chambers in Matrix of Decalcified Dentin. Arch. Surg. 1967, 94, 781–789. [Google Scholar] [CrossRef] [PubMed]
  3. Minetti, E.; Palermo, A.; Ferrante, F.; Schmitz, J.H.; Lung Ho, H.K.; Dih Hann, S.N.; Giacometti, E.; Gambardella, U.; Contessi, M.; Celko, M.; et al. Autologous Tooth Graft after Endodontical Treated Used for Socket Preservation: A Multicenter Clinical Study. Appl. Sci. 2019, 9, 5396. [Google Scholar] [CrossRef]
  4. Kim, Y.-K.; Lee, J.; Um, I.-W.; Kim, K.-W.; Murata, M.; Akazawa, T.; Mitsugi, M. Tooth-Derived Bone Graft Material. J. Korean Assoc. Oral Maxillofac. Surg. 2013, 39, 103–111. [Google Scholar] [CrossRef]
  5. Bessho, K.; Tanaka, N.; Matsumoto, J.; Tagawa, T.; Murata, M. Human Dentin-Matrix-Derived Bone Morphogenetic Protein. J. Dent. Res. 1991, 70, 171–175. [Google Scholar] [CrossRef]
  6. Pelegrine, A.A.; da Costa, C.E.S.; Correa, M.E.P.; Marques, J.F.C. Clinical and Histomorphometric Evaluation of Extraction Sockets Treated with an Autologous Bone Marrow Graft. Clin. Oral Implant. Res. 2010, 21, 535–542. [Google Scholar] [CrossRef]
  7. Kumar, G.S. Orban’s Oral Histology & Embryology—E-BOOK; Elsevier Health Sciences: Amsterdam, The Netherlands, 2015; ISBN 978-81-312-4505-7. [Google Scholar]
  8. Glimcher, M. Bone: Nature of the Calcium Phosphate Crystals and Cellular, Structural, and Physical Chemical Mechanisms in Their Formation. Rev. Mineral. Geochem. 2006, 64, 223–282. [Google Scholar] [CrossRef]
  9. Hedenbjörk-Lager, A.; Hamberg, K.; Pääkkönen, V.; Tjäderhane, L.; Ericson, D. Collagen Degradation and Preservation of MMP-8 Activity in Human Dentine Matrix after Demineralization. Arch. Oral Biol. 2016, 68, 66–72. [Google Scholar] [CrossRef]
  10. Li, P.; Zhu, H.; Huang, D. Autogenous DDM versus Bio-Oss Granules in GBR for Immediate Implantation in Periodontal Postextraction Sites: A Prospective Clinical Study. Clin. Implant. Dent. Relat. Res. 2018, 20, 923–928. [Google Scholar] [CrossRef]
  11. Dequeker, J.; Mohan, S.; Finkelman, R.D.; Aerssens, J.; Baylink, D.J. Generalized Osteoarthritis Associated with Increased Insulin-like Growth Factor Types I and II and Transforming Growth Factor Beta in Cortical Bone from the Iliac Crest. Possible Mechanism of Increased Bone Density and Protection against Osteoporosis. Arthritis Rheum. 1993, 36, 1702–1708. [Google Scholar] [CrossRef]
  12. Kim, Y.-K.; Lee, J.K.; Kim, K.-W.; Um, I.-W.; Murata, M. Healing Mechanism and Clinical Application of Autogenous Tooth Bone Graft Material. In Advances in Biomaterials Science and Biomedical Applications; BoD—Books on Demand: Norderstedt, Germany, 2013; ISBN 978-953-51-1051-4. [Google Scholar]
  13. Inchingolo, F.; Tatullo, M.; Marrelli, M.; Inchingolo, A.M.; Scacco, S.; Inchingolo, A.D.; Dipalma, G.; Vermesan, D.; Abbinante, A.; Cagiano, R. Trial with Platelet-Rich Fibrin and Bio-Oss Used as Grafting Materials in the Treatment of the Severe Maxillar Bone Atrophy: Clinical and Radiological Evaluations. Eur. Rev. Med. Pharmacol. Sci. 2010, 14, 1075–1084. [Google Scholar]
  14. ISTOLOGIA di Monesi. Available online: http://www.piccin.it/it/istologia/2355-istologia-di-monesi-9788829928132.html (accessed on 27 March 2023).
  15. Kirkham, J.; Brookes, S.J.; Shore, R.C.; Bonass, W.A.; Smith, D.A.; Wallwork, M.L.; Robinson, C. Atomic Force Microscopy Studies of Crystal Surface Topology during Enamel Development. Connect. Tissue Res. 1998, 38, 91–100; discussion 139–145. [Google Scholar] [CrossRef]
  16. Minetti, E.; Giacometti, E.; Gambardella, U.; Contessi, M.; Ballini, A.; Marenzi, G.; Celko, M.; Mastrangelo, F. Alveolar Socket Preservation with Different Autologous Graft Materials: Preliminary Results of a Multicenter Pilot Study in Human. Materials 2020, 13, 1153. [Google Scholar] [CrossRef]
  17. Butler, W.T.; Ritchie, H. The Nature and Functional Significance of Dentin Extracellular Matrix Proteins. Int. J. Dev. Biol. 1995, 39, 169–179. [Google Scholar]
  18. Dozza, B.; Lesci, I.G.; Duchi, S.; Della Bella, E.; Martini, L.; Salamanna, F.; Falconi, M.; Cinotti, S.; Fini, M.; Lucarelli, E.; et al. When Size Matters: Differences in Demineralized Bone Matrix Particles Affect Collagen Structure, Mesenchymal Stem Cell Behavior, and Osteogenic Potential. J. Biomed. Mater. Res. A 2017, 105, 1019–1033. [Google Scholar] [CrossRef]
  19. Almushayt, A.; Narayanan, K.; Zaki, A.E.; George, A. Dentin Matrix Protein 1 Induces Cytodifferentiation of Dental Pulp Stem Cells into Odontoblasts. Gene Ther. 2006, 13, 611–620. [Google Scholar] [CrossRef]
  20. Schmidt-Schultz, T.H.; Schultz, M. Intact Growth Factors Are Conserved in the Extracellular Matrix of Ancient Human Bone and Teeth: A Storehouse for the Study of Human Evolution in Health and Disease. Biol. Chem. 2005, 386, 767–776. [Google Scholar] [CrossRef]
  21. Cassidy, N.; Fahey, M.; Prime, S.S.; Smith, A.J. Comparative Analysis of Transforming Growth Factor-Beta Isoforms 1-3 in Human and Rabbit Dentine Matrices. Arch. Oral Biol. 1997, 42, 219–223. [Google Scholar] [CrossRef]
  22. Finkelman, R.D.; Mohan, S.; Jennings, J.C.; Taylor, A.K.; Jepsen, S.; Baylink, D.J. Quantitation of Growth Factors IGF-I, SGF/IGF-II, and TGF-Beta in Human Dentin. J. Bone Miner. Res. 1990, 5, 717–723. [Google Scholar] [CrossRef]
  23. Patel, Z.S.; Young, S.; Tabata, Y.; Jansen, J.A.; Wong, M.E.K.; Mikos, A.G. Dual Delivery of an Angiogenic and an Osteogenic Growth Factor for Bone Regeneration in a Critical Size Defect Model. Bone 2008, 43, 931–940. [Google Scholar] [CrossRef]
  24. Kanazirski, N.; Kanazirska, P. Auto-Tooth Bone Graft Material for Reconstruction of Bone Defects in the Oral Region: Case Reports. Folia Med. 2022, 64, 162–168. [Google Scholar] [CrossRef] [PubMed]
  25. Nakashima, M. Bone Morphogenetic Proteins in Dentin Regeneration for Potential Use in Endodontic Therapy. Cytokine Growth Factor Rev. 2005, 16, 369–376. [Google Scholar] [CrossRef] [PubMed]
  26. Urist, M.R.; Strates, B.S. Bone Morphogenetic Protein. J. Dent. Res. 1971, 50, 1392–1406. [Google Scholar] [CrossRef] [PubMed]
  27. Rijal, G.; Shin, H.-I. Human Tooth-Derived Biomaterial as a Graft Substitute for Hard Tissue Regeneration. Regen. Med. 2017, 12, 263–273. [Google Scholar] [CrossRef] [PubMed]
  28. Tang, G.; Liu, Z.; Liu, Y.; Yu, J.; Wang, X.; Tan, Z.; Ye, X. Recent Trends in the Development of Bone Regenerative Biomaterials. Front. Cell Dev. Biol. 2021, 9, 665813. [Google Scholar] [CrossRef]
  29. Hazballa, D.; Inchingolo, A.; Inchingolo, A.; Malcangi, G.; Santacroce, L.; Minetti, E.; Di Venere, D.; Limongelli, L.; Bordea, R.; Scarano, A.; et al. The Effectiveness of Autologous Demineralized Tooth Graft for the Bone Ridge Preservation: A Systematic Review of the Literature. J. Biol. Regul. Homeost. Agents 2021, 35, 283–294. [Google Scholar] [CrossRef]
  30. Bono, N.; Tarsini, P.; Candiani, G. Demineralized Dentin and Enamel Matrices as Suitable Substrates for Bone Regeneration. JABFM 2017, 15, 236–243. [Google Scholar] [CrossRef]
  31. Inchingolo, A.M.; Patano, A.; Di Pede, C.; Inchingolo, A.D.; Palmieri, G.; de Ruvo, E.; Campanelli, M.; Buongiorno, S.; Carpentiere, V.; Piras, F.; et al. Autologous Tooth Graft: Innovative Biomaterial for Bone Regeneration. Tooth Transformer® and the Role of Microbiota in Regenerative Dentistry. A Systematic Review. J. Funct. Biomater. 2023, 14, 132. [Google Scholar] [CrossRef]
  32. Caruso, S.; Bernardi, S.; Pasini, M.; Giuca, M.R.; Docimo, R.; Continenza, M.A.; Gatto, R. The Process of Mineralisation in the Development of Human Tooth. Eur. J. Paediatr. Dent. 2016, 17, 322–326. [Google Scholar]
  33. Chen, W.-C.; Chung, C.-H.; Lu, Y.-C.; Wu, M.-H.; Chou, P.-H.; Yen, J.-Y.; Lai, Y.-W.; Wang, G.-S.; Liu, S.-C.; Cheng, J.-K.; et al. BMP-2 Induces Angiogenesis by Provoking Integrin A6 Expression in Human Endothelial Progenitor Cells. Biochem. Pharmacol. 2018, 150, 256–266. [Google Scholar] [CrossRef]
  34. Huh, J.-B.; Yang, J.-J.; Choi, K.-H.; Bae, J.; Lee, J.-Y.; Kim, S.-E.; Shin, S.-W. Effect of RhBMP-2 Immobilized Anorganic Bovine Bone Matrix on Bone Regeneration. Int. J. Mol. Sci. 2015, 16, 16034–16052. [Google Scholar] [CrossRef]
  35. Kakuta, A.; Tanaka, T.; Chazono, M.; Komaki, H.; Kitasato, S.; Inagaki, N.; Akiyama, S.; Marumo, K. Effects of Micro-Porosity and Local BMP-2 Administration on Bioresorption of β-TCP and New Bone Formation. Biomater. Res. 2019, 23, 12. [Google Scholar] [CrossRef]
  36. Nampo, T.; Watahiki, J.; Enomoto, A.; Taguchi, T.; Ono, M.; Nakano, H.; Yamamoto, G.; Irie, T.; Tachikawa, T.; Maki, K. A New Method for Alveolar Bone Repair Using Extracted Teeth for the Graft Material. J. Periodontol. 2010, 81, 1264–1272. [Google Scholar] [CrossRef]
  37. Andrade, C.; Camino, J.; Nally, M.; Quirynen, M.; Martínez, B.; Pinto, N. Combining Autologous Particulate Dentin, L-PRF, and Fibrinogen to Create a Matrix for Predictable Ridge Preservation: A Pilot Clinical Study. Clin. Oral Investig. 2020, 24, 1151–1160. [Google Scholar] [CrossRef]
  38. Koga, T.; Minamizato, T.; Kawai, Y.; Miura, K.; I, T.; Nakatani, Y.; Sumita, Y.; Asahina, I. Bone Regeneration Using Dentin Matrix Depends on the Degree of Demineralization and Particle Size. PLoS ONE 2016, 11, e0147235. [Google Scholar] [CrossRef]
  39. Graham, L.; Cooper, P.R.; Cassidy, N.; Nor, J.E.; Sloan, A.J.; Smith, A.J. The Effect of Calcium Hydroxide on Solubilisation of Bio-Active Dentine Matrix Components. Biomaterials 2006, 27, 2865–2873. [Google Scholar] [CrossRef]
  40. Smith, A.J.; Tobias, R.S.; Plant, C.G.; Browne, R.M.; Lesot, H.; Ruch, J.V. In Vivo Morphogenetic Activity of Dentine Matrix Proteins. J. Biol. Buccale 1990, 18, 123–129. [Google Scholar]
  41. Cai, Y.; Liu, Y.; Yan, W.; Hu, Q.; Tao, J.; Zhang, M.; Shi, Z.; Tang, R. Role of Hydroxyapatite Nanoparticle Size in Bone Cell Proliferation. J. Mater. Chem. 2007, 17, 3780–3787. [Google Scholar] [CrossRef]
  42. Teruel, J.d.D.; Alcolea, A.; Hernández, A.; Ruiz, A.J.O. Comparison of Chemical Composition of Enamel and Dentine in Human, Bovine, Porcine and Ovine Teeth. Arch. Oral Biol. 2015, 60, 768–775. [Google Scholar] [CrossRef]
  43. Balhuc, S.; Campian, R.; Labunet, A.; Negucioiu, M.; Buduru, S.; Kui, A. Dental Applications of Systems Based on Hydroxyapatite Nanoparticles—An Evidence-Based Update. Crystals 2021, 11, 674. [Google Scholar] [CrossRef]
  44. Minetti, E.; Berardini, M.; Trisi, P. A New Tooth Processing Apparatus Allowing to Obtain Dentin Grafts for Bone Augmentation: The Tooth Transformer. Open Dent. J. 2019, 13, 6–14. [Google Scholar] [CrossRef]
  45. Reith, E.J. The Early Stage of Amelogenesis as Observed in Molar Teeth of Young Rats. J. Ultrastruct. Res. 1967, 17, 503–526. [Google Scholar] [CrossRef] [PubMed]
  46. Scheven, B.a.A.; Marshall, D.; Aspden, R.M. In Vitro Behaviour of Human Osteoblasts on Dentin and Bone. Cell Biol. Int. 2002, 26, 337–346. [Google Scholar] [CrossRef] [PubMed]
  47. Tabatabaei, F.S.; Tatari, S.; Samadi, R.; Moharamzadeh, K. Different Methods of Dentin Processing for Application in Bone Tissue Engineering: A Systematic Review. J. Biomed. Mater. Res. A 2016, 104, 2616–2627. [Google Scholar] [CrossRef]
  48. Marshall, G.W.; Marshall, S.J.; Kinney, J.H.; Balooch, M. The Dentin Substrate: Structure and Properties Related to Bonding. J. Dent. 1997, 25, 441–458. [Google Scholar] [CrossRef]
  49. Goldberg, M.; Kulkarni, A.B.; Young, M.; Boskey, A. Dentin: Structure, Composition and Mineralization. Front. Biosci. Elite Ed. 2011, 3, 711–735. [Google Scholar] [CrossRef]
  50. Iasella, J.M.; Greenwell, H.; Miller, R.L.; Hill, M.; Drisko, C.; Bohra, A.A.; Scheetz, J.P. Ridge Preservation with Freeze-Dried Bone Allograft and a Collagen Membrane Compared to Extraction Alone for Implant Site Development: A Clinical and Histologic Study in Humans. J. Periodontol. 2003, 74, 990–999. [Google Scholar] [CrossRef]
  51. Younger, E.M.; Chapman, M.W. Morbidity at Bone Graft Donor Sites. J. Orthop. Trauma 1989, 3, 192–195. [Google Scholar] [CrossRef]
  52. Sittitavornwong, S.; Gutta, R. Bone Graft Harvesting from Regional Sites. Oral Maxillofac. Surg. Clin. N. Am. 2010, 22, 317–330. [Google Scholar] [CrossRef]
  53. Stevenson, S. Biology of Bone Grafts. Orthop. Clin. N. Am. 1999, 30, 543–552. [Google Scholar] [CrossRef]
  54. Elgali, I.; Omar, O.; Dahlin, C.; Thomsen, P. Guided Bone Regeneration: Materials and Biological Mechanisms Revisited. Eur. J. Oral Sci. 2017, 125, 315–337. [Google Scholar] [CrossRef]
  55. Gideon Hallel, I.B. A Novel Procedure to Process Extracted Teeth for Immediate Grafting of Autogenous Dentin. J. Interdiscipl. Med. Dent. Sci. 2014, 2, 6. [Google Scholar] [CrossRef]
  56. Moharamzadeh, K.; Freeman, C.; Blackwood, K. Processed Bovine Dentine as a Bone Substitute. Br. J. Oral Maxillofac. Surg. 2008, 46, 110–113. [Google Scholar] [CrossRef]
  57. Kim, K.-W. Bone Induction by Demineralized Dentin Matrix in Nude Mouse Muscles. Maxillofac. Plast. Reconstr. Surg. 2014, 36, 50–56. [Google Scholar] [CrossRef]
  58. Kravchik, M.V.; Zolotenkova, G.V.; Grusha, Y.O.; Pigolkin, Y.I.; Fettser, E.I.; Zolotenkov, D.D.; Gridina, N.V.; Badyanova, L.V.; Alexandrov, A.A.; Novikov, I.A. Age-Related Changes in Cationic Compositions of Human Cranial Base Bone Apatite Measured by X-Ray Energy Dispersive Spectroscopy (EDS) Coupled with Scanning Electron Microscope (SEM). Biometals 2022, 35, 1077–1094. [Google Scholar] [CrossRef]
  59. Okubo, N.; Ishikawa, M.; Shakya, M.; Hosono, H.; Maehara, O.; Ohkawara, T.; Ohnishi, S.; Akazawa, T.; Murata, M. Autograft of Demineralized Dentin Matrix Prepared Immediately after Extraction for Horizontal Bone Augmentation of the Anterior Atrophic Maxilla: A First Case of Non-Vital Tooth-Derived Dentin. J. Hard Tissue Biol. 2022, 31, 47–54. [Google Scholar] [CrossRef]
  60. Cervera-Maillo, J.M.; Morales-Schwarz, D.; Morales-Melendez, H.; Mahesh, L.; Calvo-Guirado, J.L. Autologous Tooth Dentin Graft: A Retrospective Study in Humans. Medicina 2021, 58, 56. [Google Scholar] [CrossRef]
Figure 1. Electron microscopy (5000×) surface of a non-demineralized dentin granule. The surface is rough, dirty, and full of debris, and the dental tubules are not clean or closed.
Figure 1. Electron microscopy (5000×) surface of a non-demineralized dentin granule. The surface is rough, dirty, and full of debris, and the dental tubules are not clean or closed.
Jfb 14 00272 g001
Figure 2. Electron microscopy (5000×) surface of a demineralized dentin after the Tooth Transformer treatment. The surface is cleaner and smoother than non-demineralized dentin.
Figure 2. Electron microscopy (5000×) surface of a demineralized dentin after the Tooth Transformer treatment. The surface is cleaner and smoother than non-demineralized dentin.
Jfb 14 00272 g002
Figure 3. Electron microscopy (5000×) surface of a bone sample. The surface is smooth, flat, and free of irregularities and dental tubules.
Figure 3. Electron microscopy (5000×) surface of a bone sample. The surface is smooth, flat, and free of irregularities and dental tubules.
Jfb 14 00272 g003
Table 1. Bone and dentin HA crystal dimensions.
Table 1. Bone and dentin HA crystal dimensions.
HA CrystalsBoneDentin
Length20–60 nm80 ± 12 (nm)
Width5–20 nm40 ± 10 nm
Thickness1, 2/3 nm3–4 nm
Table 2. Comparison between group A and group C. Eleven samples were analyzed by elemental analysis, and atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared. The p-value was indicated for each comparison, and all comparisons were found to be statistically different. Analyze: p > 0.05—no statistical significance value. If p < 0.05—the hypothesis is wrong.
Table 2. Comparison between group A and group C. Eleven samples were analyzed by elemental analysis, and atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared. The p-value was indicated for each comparison, and all comparisons were found to be statistically different. Analyze: p > 0.05—no statistical significance value. If p < 0.05—the hypothesis is wrong.
Group A vs. Group C
DatasetCNon-treated dentinBoneONon-treated dentinBoneCaNon-treated dentinBonePNon-treated dentinBone
Sample size1111111111111111
Average24.020044.971049.980013.354016.56009.92608.59004.0280
Standard deviation4.350011.76804.65001.74006.12006.74701.91002.7700
T5.538424.46682.41544.4969
Degree of freedom20202020
p-value 0.000020.000000000020.02540.0002
Table 3. Comparison between group B and group C. Eleven samples were analyzed by elemental analysis, and atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared. The p-value was indicated for each comparison, and all are without any statistical differences. Analyze: p > 0.05—no statistical significance value. If p < 0.05—the hypothesis is wrong.
Table 3. Comparison between group B and group C. Eleven samples were analyzed by elemental analysis, and atomic percentages of C (carbon), O (oxygen), Ca (calcium), and P (phosphorus) were individually analyzed and compared. The p-value was indicated for each comparison, and all are without any statistical differences. Analyze: p > 0.05—no statistical significance value. If p < 0.05—the hypothesis is wrong.
Group B vs. Group C
DatasetCTreated dentinBoneOTreated dentinBoneCaTreated dentinBonePTreated dentinBone
Sample size1111111111111111
Average60.020044.971026.060013.35408.59009.92605.04004.0280
Standard deviation3.790011.76803.06001.74001.37006.74700.60002.7700
T4.037111.97150.64361.1842
Degree of freedom20202020
p-value 0.00060.000020.52710.2502
Table 4. Bone and dentin differences at different scales.
Table 4. Bone and dentin differences at different scales.
BoneDentin
Tissue scale (millimeters to micrometers)Bone is an organic matrix of connective tissue composed of cells, fibers, and inorganic matrix ground substance. The cells control the initial production of the mineralized tissue.Teeth are composed of cells, an organic matrix, and an inorganic matrix.
Microstructure scale (micrometers)Individual struts (trabeculae) present in the marrow connecting the bone structure, thin plates (lamellae) in the cortical bone, and bone developed around blood veins are all structural units of bone (termed osteons).Dentinal tubules and the intratubular dentin that surrounds the dentin-forming odontoblasts are structural units of the tooth.
Ultrastructural scale (nanometers)Tissue components are distinct in the mineral crystals and the organic matrix. The bone’s organic matrix mostly comprises of a fibrous protein, collagen, and trace amounts of other non-collagenous proteins.Collagen is the primary organic constituent of dentin and cementum. However, there is no collagen present in enamel. An equivalent of the mineral hydroxyapatite is the mineral that reinforces dentin matrices and is also a major constituent of enamel.
Table 5. Different mineralization phases between the bone and dentin.
Table 5. Different mineralization phases between the bone and dentin.
Mineralization PhaseBoneDentin
First phaseOsteoblasts secrete organic matrix (in particular collagen and non-collagenic proteins) and bone vesicle matrixOdontoblasts secrete collagen and non-collagenic proteins.
Second phaseAlso termed the vesicular phase, vesicles—which have accumulated calcium and phosphorus—begin to nucleate the calcium and phosphorus salts. The crystalline structures grow due to the entry of phosphorus and calcium ions, resulting in the breakage of the vesicle membrane.Nucleation of vesicles -which have accumulated calcium and phosphorus. The osteoblast elongates a cytoplasmatic process into the dentinal tubules.
Third phaseTermed the fibrillar phase, the hydroxyapatite crystals further increase in the bone extracellular matrix and form the so-called matrix nodules that are associated with the organic matrix of the osteoid substance, in particular with collagen fibrils. It is precisely on the collagen fibers that the hydroxyapatite crystals are deposited, and therefore we speak, in fact, of “collagen mineralization”.Growth of the crystals occurs. The crystals linked to the collagen fibrils are arranged in rows that conform to the 64 nm striation pattern, with their long axes paralleling the fibril long axes. The progress of general calcification is gradual. Dentin apatite crystals are similar to those found in the bone and cementum, and are 300 times smaller in size than those made in the enamel.
Table 6. List of proteins present in the bone and dentin.
Table 6. List of proteins present in the bone and dentin.
Main Bone ProteinsMain Dentin Proteins
GLA proteinGLA protein
OPNOPN
OsteonectinCbfa 1 RUNX2
ProteoglycansBMP
BMPIGI I and IGF II
PDGFTGF-b 19
FGFDSP
IGFDGP, DPP
Lysyl oxidaseType I, III, V collagen
TRAMP
BSP
Type I, III, V collagen
Table 7. The quantity of donor bone sites calculated from Somsak/Rajesh analysis.
Table 7. The quantity of donor bone sites calculated from Somsak/Rajesh analysis.
Donor SitesSize of Corticocancellous BlockVolume
Symphysis20.9 × 9.9 × 6.9 mm34.71
Ascending ramus37.6 × 33.17 × 22.48 × 9.15 mm32.36
Lateral ramus1.3 cm × 3 cm3Not applicable
Coronoid process18 × 17 × 5 mm3Not applicable
Zygomatic buttress1.5 × 2.0 mm3Not applicable
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Minetti, E.; Palermo, A.; Malcangi, G.; Inchingolo, A.D.; Mancini, A.; Dipalma, G.; Inchingolo, F.; Patano, A.; Inchingolo, A.M. Dentin, Dentin Graft, and Bone Graft: Microscopic and Spectroscopic Analysis. J. Funct. Biomater. 2023, 14, 272. https://doi.org/10.3390/jfb14050272

AMA Style

Minetti E, Palermo A, Malcangi G, Inchingolo AD, Mancini A, Dipalma G, Inchingolo F, Patano A, Inchingolo AM. Dentin, Dentin Graft, and Bone Graft: Microscopic and Spectroscopic Analysis. Journal of Functional Biomaterials. 2023; 14(5):272. https://doi.org/10.3390/jfb14050272

Chicago/Turabian Style

Minetti, Elio, Andrea Palermo, Giuseppina Malcangi, Alessio Danilo Inchingolo, Antonio Mancini, Gianna Dipalma, Francesco Inchingolo, Assunta Patano, and Angelo Michele Inchingolo. 2023. "Dentin, Dentin Graft, and Bone Graft: Microscopic and Spectroscopic Analysis" Journal of Functional Biomaterials 14, no. 5: 272. https://doi.org/10.3390/jfb14050272

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop