Demineralized dentin matrix technique - a comparison of different demineralizing solutions

Abstract This study aimed to evaluate the microstructure formed after the chemical treatment of teeth, for the development of autogenous grafts from the demineralized dentin matrix (DDM) technique, in order to identify the most efficient demineralizing solution. The specimens, originating from the root and coronal portion, were submitted to ultrasonic cleaning and drying in an oven for 1h at 100 ºC. Then, the density was determined by Archimedes' principle for each specimen, using distilled water as immersion liquid. The samples were separated into five groups: Control group: negative control, Distilled water;EDTA group: positive control, trisodium EDTA; NaOCl group: 2.5% sodium hypochlorite; HCl-0.6M group: 0.6M hydrochloric acid; and H2O2/H2SO4 group: hydrogen peroxide and sulfuric acid. Each specimen was immersed for 1h in the corresponding group descaling solution at 60 ºC. Subsequently, the mass loss and density of the treated specimens were determined by Archimedes' principle. Ultimately, the specimens of each group were characterized by microtomography, Scanning Electron Microscopy, and Energy Dispersive Spectrometry X-ray (SEM-EDS). The results demonstrated that the H2O2/H2SO4 solution allowed the formation of interconnected micropores, suggesting better pore structures for application in scaffolds, when compared to the other studied solutions.

Resumo Este estudo teve como objetivo avaliar a microestrutura formada após o tratamento químico em dentes, para o desenvolvimento de enxertos autógenos a partir da técnica de matriz de dentina desmineralizada (DDM), a fim de identificar a solução desmineralizante mais eficiente. Os espécimes, provenientes da raiz e porção coronal, foram submetidos à limpeza ultrassônica e secagem em estufa por 1h a 100 ºC. Em seguida, a densidade foi determinada pelo princípio de Arquimedes para cada espécime, utilizando água destilada como líquido de imersão. As amostras foram separadas em cinco grupos: Controle: controle negativo, Água destilada; EDTA: controle positivo, EDTA trissódico; NaOCl: hipoclorito de sódio 2,5%; HCl-0.6M: ácido clorídrico 0,6M; e H2O2/H2SO4: peróxido de hidrogênio e ácido sulfúrico. Cada espécime foi imerso por 1h na solução descalcificante de grupo correspondente a 60 ºC. Posteriormente, a perda de massa e a densidade dos espécimes tratados foram determinadas pelo princípio de Arquimedes. Por fim, os espécimes de cada grupo foram caracterizados por microtomografia, microscopia eletrônica de varredura e espectrometria de energia dispersiva de raios-X (SEM-EDS). Os resultados demonstraram que a solução H2O2/H2SO4 permitiu a formação de microporos interligados, sugerindo melhores estruturas de poros para aplicação em scaffolds, quando comparada às demais soluções estudadas.

Postulated release profile of recombinant human bone morphogenetic protein-2 (rhBMP-2) from demineralized dentin matrix

Demineralized dentin matrix (DDM) has been used as a recombinant human bone morphogenetic protein-2 (rhBMP-2) carrier in many clinical trials. To optimize the clinical safety and efficacy of rhBMP-2 with DDM, efforts have been made to improve the delivery of rhBMP-2 by 1) lowering the administered dose, 2) localizing the protein, and 3) prolonging its retention time at the action site as well as the bone forming capacity of the carrier itself. The release profile of rhBMP-2 that is associated with endogenous BMP in dentin has been postulated according to the type of incorporation, which is attributed to the loosened interfibrillar space and nanoporous dentinal tubule pores. Physically adsorbed and modified, physically entrapped rhBMP-2 is sequentially released from the DDM surface during the early stage of implantation. As DDM degradation progresses, the loosened interfibrillar space and enlarged dentinal tubules release the entrapped rhBMP-2. Finally, the endogenous BMP in dentin is released with osteoclastic dentin resorption. According to the postulated release profile, DDM can therefore be used in a controlled manner as a sequential delivery scaffold for rhBMP-2, thus sustaining the rhBMP-2 concentration for a prolonged period due to localization. In addition, we attempted to determine how to lower the rhBMP-2 concentration to 0.2 mg/mL, which is lower than the approved 1.5 mg/mL.

Research on the surface structure of a dentin matrix with complete demineralization and incomplete demineralization and the osteogenic property promotion of human periodontal ligament cells / 口腔疾病防治

Objective@# To analyze the different fabrication methods and surface structure of treated dentin matrix (TDM) and demineralized dentin matrix (DDM) and their diverse function on promoting the proliferation and osteogenic differential capability of human periodontal ligament cells (hPDLCs). This study provides a preliminary basis for the treatment of periodontal bone defects with bone substitutes from teeth.@*Methods@#TDM was made from human dentin matrices and demineralized incompletely by soaking in different concentrations of ethylene diamine tetra-acetic while DDM was made of human dentin matrices and demineralized completely by soaking in a hydrochloric acid solution followed by observation via SEM. The liquid extracts of TDM and DDM were collected according to the protocol of the International Standardization Organization (ISO 10993). Then, hPDLCs were divided into the following three groups: the TDM group (liquid extracts of TDM), the DDM group (liquid extracts of DDM), the control group (a-modified eagle medium with 10% fetal bovine serum), hPDLCs were cultured with liquid extracts of TDM or DDM, or a-modified eagle medium with 10% FBS). hPDLC proliferation was detected by a Cell Counting Kit-8 (CCK-8). The alkaline phosphatase (ALP) expression and calcified nodules of hPDLCs were tested.@*Results @#TDM obtained a preferable surface structure compared to DDM due to more sufficiently exposed dentinal tubules and looser fiber bundles of the intertubular and peritubular dentin. Both TDM and DDM promoted the proliferation of hPDLCs compared with the control group, and the proliferation of hPDLCs was significantly greater in the TDM group compared to the DDM group (F = 36.480, P < 0.05). The ALP activity of hPDLCs in the TDM group was higher than the DDM group. After a 14-day osteogenic induction, Alizarin red staining mineral nodes were observed in both groups; however, the TDM group displayed more calcified nodules than the DDM group.@*Conclusion@#The advantages of TDM including the surface structure, proliferation and osteogenic differentiation of hPDLCs, are more prominent than those of DDM, suggesting that TDM is a potential promising bone graft substitute in periodontal regeneration.

Experimental study of demineralized dentin matrix on osteoinduction and related cells identification / 华西口腔医学杂志

OBJECTIVE@#The aim of this study was to explore the theoretical framework of cells and the forms of osteogenesis in the mechanism by which demineralized dentin matrix (DDM) induces osteogenesis.@*METHODS@#A total of 24 New Zealand rabbits were used in this study. A total of 4 erector spinae bags were created in each animal. A total of 3 erector spinae bags were implanted with DDM by random selection, whereas the remaining one erector spinae bag was not implanted with DDM. The rabbits were sacrificed after 1, 2, 3, 4, 8, 12, 16, and 20 weeks, and the samples were obtained. The samples were examined by hematoxylin-eosin (HE), tartrate-resistant acid phosphatase (TRAP), and immunohistochemical staining to identify the mesenchymal stem cells, osteoblasts, chondrocytes, and osteoclasts.@*RESULTS@#The results of HE staining showed that in the third week, cartilage- and bone-like matrices, as well as the osteoblast-like cells, were observed. The results of immunohistochemical staining showed that the expressions of CD44, alkaline phosphatase (ALP), and collagen Ⅱ were statistically significant 
(P<0.05).@*CONCLUSIONS@#DDM has good histocompatibility and osteoinduction. In addition, induced ectopic osteogenesis mode mainly occurs in the endochondral bone.

Demineralized dentin matrix combined with recombinant human bone morphogenetic protein-2 in rabbit calvarial defects

OBJECTIVES: The aim of this study was to compare the osteogenic effects of demineralized dentin matrix (DDM) combined with recombinant human bone morphogenetic protein-2 (rhBMP-2) in rabbit calvarial defects with DDM and anorganic bovine bone (ABB) combined with rhBMP-2. MATERIALS AND METHODS: Four round defects with 8-mm diameters were created in each rabbit calvaria. Each defect was treated with one of the following: 1) DDM, 2) ABB/rhBMP-2, or 3) DDM/rhBMP-2. The rhBMP-2 was combined with DDM and ABB according to a stepwise dry and dip lyophilizing protocol. Histological and microcomputed tomography (µCT) analyses were performed to measure the amount of bone formation and bone volume after 2- and 8-week healing intervals. RESULTS: Upon histological observation at two weeks, the DDM and ABB/rhBMP-2 groups showed osteoconductive bone formation, while the DDM/rhBMP-2 group showed osteoconductive and osteoinductive bone formation. New bone formation was higher in DDM/rhBMP-2, DDM and ABB decreasing order. The amounts of bone formation were very similar at two weeks; however, at eight weeks, the DDM/rhBMP-2 group showed a two-fold greater amount of bone formation compared to the DDM and ABB/rhBMP-2 groups. The µCT analysis showed markedly increased bone volume in the DDM/rhBMP-2 group at eight weeks compared with that of the DDM group. Notably, there was a slight decrease in bone volume in the ABB/rhBMP-2 group at eight weeks. There were no significant differences among the DDM, ABB/rhBMP-2, and DDM/rhBMP-2 groups at two or eight weeks. CONCLUSION: Within the limitations of this study, DDM appears to be a suitable carrier for rhBMP-2 in orthotopic sites.

Histologic study of bone-forming capacity on polydeoxyribonucleotide combined with demineralized dentin matrix / 대한악안면성형재건외과학회지

BACKGROUND: This study examined the osteoinductive activity of demineralized dentin matrix (DDM) from human and polydeoxyribonucleotide (PDRN) for nude mice. METHODS: Twenty healthy nude mice, weighing about 15~20 g, were used for the study. DDM from human and PDRN were prepared and implanted subcutaneously into the dorsal portion of the nude mice. The nude mice were sacrificed at 1, 2, and 4 weeks after grafting and evaluated histologically by hematoxylin-eosin and Masson's trichrome staining. The specimens were also evaluated via a histomorphometric study. RESULTS: The DDM and PDRN induced new bone, osteoblasts, and fibroblasts in soft tissues. The histological findings showed bone-forming cells like osteoblasts and fibroblasts at 1, 2, and 4 weeks. New bone formation was observed in the histomorphometric study. In particular, the ratio of new bone formation was the highest at 2 weeks compared with the first week and fourth week. CONCLUSIONS: In this study, we showed that the PDRN used in this experimental model was able to induce bone regeneration when combined to the DDM.

Cranial bone regeneration according to different particle sizes and densities of demineralized dentin matrix in the rabbit model / 대한악안면성형재건외과학회지

BACKGROUND: The objective of this study was to place bone graft materials in cranial defects in a rabbit model and compare their bone regenerating ability according to the size and density of demineralized dentin matrix (DDM). METHODS: We selected nine healthy male rabbits that were raised under the same conditions and that weighed about 3 kg. Two circular defects 8 mm in diameter were created in each side of the cranium. The defects were grafted with DDM using four different particle sizes and densities: 0.1 mL of 0.25- to 1.0-mm particles (group 1); 0.2 mL of 0.25- to 1.0-mm particles (group 2); 0.1 mL of 1.0- to 2.0-mm particles (group 3); and 0.2 mL of 1.0- to 2.0-mm particles (group 4). After 2, 4, and 8 weeks, the rabbits were sacrificed, and bone samples were evaluated by means of histologic, histomorphometric, and quantitative RT-PCR analysis. RESULTS: In group 1, osteoblast activity and bone formation were greater than in the other three groups on histological examination. In groups 2, 3, and 4, dense connective tissue was seen around original bone even after 8 weeks. Histomorphometric analysis of representative sections in group 1 showed a higher rate of new bone formation, but the difference from the other groups was not statistically significant. RT-PCR analysis indicated a correlation between bone formation and protein (osteonectin and osteopontin) expression. CONCLUSIONS: DDM with a space between particles of 200 μm was effective in bone formation, suggesting that materials with a small particle size could reasonably be used for bone grafting.

Comparison of autogenous tooth bone graft and synthetic bone graft materials used for bone resorption around implants after crestal approach sinus lifting: a retrospective study

PURPOSE: This retrospective study compares the amount of bone resorption around implants between an autogenous tooth bone graft (AutoBT) and a synthetic bone graft after a bone-added crestally approached sinus lift with simultaneous implant placements. METHODS: In all, 37 patients participated in this study. Seventeen patients were grouped as group I and underwent an AutoBT-added sinus lift using the crestal approach. The remaining 20 patients were grouped as group II and underwent synthetic bone grafting. Both groups received the implant placements simultaneously. Of the 37 participating patients, only 22 patients were included in the final results: Eleven patients of group I and 11 patients of group II. Before the surgery, the distance from the alveolar crest to the sinus floor was measured using panoramic radiography. After the surgery, the distance was measured again from the neck of the implant thread to the most superior border of the added graft materials. Then, the amount of sinus lift was calculated by comparing the two panoramic radiographs. After a year, a panoramic radiograph was taken to calculate the resorption of the bone graft material from the radiograph that was taken after the surgery. The significance of the resorption amount between the two types of graft materials was statistically analyzed. RESULTS: The bone height was increased to an average of 4.89 mm in group I and 6.22 mm in group II. The analysis of panoramic radiographs 1 year after the surgery showed an average bone resorption of 0.76 mm and 0.53 mm, respectively. However, the degree of lifting (P=0.460) and the amount of bone-grafted material resorption (P=0.570) showed no statistically significant difference. CONCLUSIONS: Based on this limited study, AutoBT can be considered a good alternative bone graft to a synthetic bone graft in a bone-added sinus lift, when extraction is necessary prior to the surgery.

Tooth-derived bone graft material

With successful extraction of growth factors and bone morphogenic proteins (BMPs) from mammalian teeth, many researchers have supported development of a bone substitute using tooth-derived substances. Some studies have also expanded the potential use of teeth as a carrier for growth factors and stem cells. A broad overview of the published findings with regard to tooth-derived regenerative tissue engineering technique is outlined. Considering more than 100 published papers, our team has developed the protocols and techniques for processing of bone graft material using extracted teeth. Based on current studies and studies that will be needed in the future, we can anticipate development of scaffolds, homogenous and xenogenous tooth bone grafts, and dental restorative materials using extracted teeth.

Analysis of Organic Components and Osteoinductivity in Autogenous Tooth Bone Graft Material

Study of bone healing pattern in extraction socket after application of demineralized dentin matrix material

INTRODUCTION: Research on dental bone graft material has been actively conducted. Recently, demineralized dentin matrix material has been developed and introduced. This study examined the effect of demineralized dentin matrix material on bone healing. SUBJECTS AND METHODS: The patients who received no treatment after extraction were used as the control group and patients who underwent demineralized dentin matrix material application in the extraction socket after extraction were used as the experimental group. Panorama radiography was performed at the baseline and at 3.5 months after graft material placement and CT was taken at 3.5 months after graft material placement for a radiologic evaluation. Bony tissue specimens were collected from the alveolar crest in the middle of the extraction socket using a 2 mm trephine bur after 3.5 months for the histology and hostomorphometric study. RESULTS: 1. On the panoramic view, a higher bone density was observed in the subject group. 2. On the panoramic view, the bone density increased significantly in the extraction socket, from the baseline to 3.5 months: a 7 and 10 gray-level scale was observed in the control and experimental group, respectively (P<0.05). 3. The CT view evaluation at 3.5 months revealed significantly higher bone density in the subject group than the control group (P<0.05). 4. The histological findings showed more active new bone and lamellar bone formation in the subject group. Dentin with osteoinduction ability and enamel with osteoconduction ability appeared. 5. On histomorphometric analysis, the subject group showed significantly more new bone, lamellar bone area and lower soft tissue area (P<0.05). The difference between the groups was significant (P<0.05). CONCLUSION: Bone healing was improved after the application of demineralized dentin matrix material and there was active new bone and lamellar bone formation.