Characterization of progenitor/stem cell population from human dental socket and their multidifferentiation potential.

Dental stem cells have many applications in medicine, dentistry and stem cell biology in general due to their easy accessibility and low morbidity. A common surgical manoeuvre after a tooth extraction is the dental socket curettage which is necessary to clean the alveolus and favour alveolar bone healing. This procedure can cause very low morbidity compared to bone marrow collection procedures and the collected material is normally discarded. In order to investigate if the tissue obtained by dental socket curettage after a tooth extraction was a feasible alternative source to isolate human stem cells, we isolated and characterized two different stem cell populations based on STRO-1 and CD146 expression. We were able to collect and grow cells from dental socket of vital and non-vital teeth. Both populations were proliferative, clonogenic and expressed STRO-1, CD146, CD90, NG2, PDGFR-ß, which are markers found in stem cells, presented in vitro multiline-differentiation into osteogenic, chondrogenic, and adipogenic tissue, and in vivo transplanted cells formed mineralized tissue. Interestingly, STRO-1+ clonogenic cells presented better multidifferentiation than CD146+ cells. Our results showed that mesenchymal stem cells can be isolated from the tiny tissue collected by dental socket curettage after vital and non-vital tooth extraction and suggest that STRO-1 is an important marker to be used to sort cells with multidifferentiation capacity.

Socket-shield technique: the influence of the length of the remaining buccal segment of healthy tooth structure on peri-implant bone and socket preservation. A study in dogs.

OBJECTIVE: The aim of this study was to evaluate the influence of the location and length of root pieces on buccal peri-implant bone width and socket preservation in socket shield technique. MATERIAL AND METHODS: Forty-eight dental implants (24 narrow and 24 regular platform internal hex implants) were placed in six dogs. The clinical crowns of teeth P2, P3, P4 and M1 were detached horizontally and removed from the underlying roots. Then the mesial root of each tooth was extracted and the distal root was degraded using a high-speed hand-piece with round bur, creating a concave shell of dentin cementum and periodontal ligament (PDL) connected to the buccal aspect of the socket. Remaining root fragments of different lengths were created: coronal (1/3); middle and coronal (2/3); full length (3/3). These were positioned all around the bone crest. Implants were placed at the center of the root sockets, 1-3mm deeper than the original root apex. RFA and histological evaluations were made at 4 and 12 weeks. Data underwent statistical analysis (p<0.05). RESULTS: All 48 implants osseointegrated satisfactorily. On both buccal and lingual sides, the coronal (1/3) radicular fragment was attached to the buccal bone plate by physiologic periodontal ligament with less crestal bone resorption compared with middle (2/3) and whole root (3/3) groups for narrow and standard implants. CONCLUSIONS: Within the limitations of this study, the results demonstrate that a small piece of root in the coronal part of the alveolus can protect the buccal, mesial and distal bone crest following the immediate placement of NeO narrow or NeO Standard Internal Hex implants. The thickness of peri-implant bone and the remaining root fragment together will provide a total thickness of >2mm. The technique would appear to be highly predictable, maintaining bone volume and reducing the risk of crestal bone resorption.

Ridge preservation of extraction sockets with chronic pathology using Bio-Oss® Collagen with or without collagen membrane: an experimental study in dogs.

OBJECTIVES: This study aimed to evaluate the dynamics of newly bone formation and dimensional change in diseased extraction sockets using Bio-Oss® Collagen with or without a collagen membrane. MATERIAL AND METHODS: In six beagle dogs, right and left 3rd and 4th mandibular premolars were hemisected and the distal roots were removed. Combined endodontic-periodontic lesions were induced in all sites using black silk, collagen sponge, endodontic files, and application of Porphyromonas gingivalis. After 4 months, among 4 premolars, three teeth were randomly selected per dog and allocated to the following experimental groups: Control group (no treatment but debridement), Test 1 group (only Bio-Oss® Collagen graft), and Test 2 group (Bio-Oss® Collagen graft with a collagen membrane). After 7 months from the baseline, the beagle dogs were sacrificed for histomorphometric and Micro-CT analysis. RESULTS: The vertical distance between buccal and lingual crests in the Control group (2.22 ± 0.26 mm) and Test 2 group (1.80 ± 0.16 mm) was significantly different. The socket of the Test 2 group (27.04 ± 5.25%) was occupied by a greater quantity of bone graft compared to the Test 1 group (18.49 ± 2.11%). CONCLUSION: Ridge preservation in diseased extraction sockets could compensate for buccal bone resorption by contact osteogenesis surrounding the bone graft particles at the bucco-coronal area during socket healing, and the application of a collagen membrane at the entrance of the socket is useful for preserving graft material at the coronal part of the socket.

Efeitos do PDGF-BB na taxa de proliferação e na adesão de células derivadas da granulação óssea a fragmentos radiculares / Effects of PDGF-BB on the rate of proliferation and on the adhesion of human cells derived from bone granulation tissue to root fragments

O objetivo deste estudo foi investigar o papel do fator de crescimento derivado de plaquetas-BB (PDGF-BB) na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea humana a fragmentos radiculares periodontalmente comprometidos. Na primeira etapa do estudo, foi estabelecida cultura primária de células da granulação óssea de dois pacientes adultos, sistemicamente saudáveis, não fumantes. Após a expansão celular, as células foram caracterizadas para determinação do fenótipo por meio de ensaios de viabilidade celular, MTT, ensaio de atividade de fosfatase alcalina, ensaio de mineralização e caracterização imunohistoquímica por meio de citometria de fluxo (segunda etapa). Na terceira etapa do estudo, os efeitos da adição de PDGF-BB recombinante humano na concentração de 300ng/ml na taxa de proliferação e adesão de células derivadas da granulação óssea a superfícies radiculares periodontalmente comprometidas foram investigados. A taxa de proliferação celular estimulada pelo PDGF-BB (grupo teste) ou pelo meio de cultura (grupo controle) foi investigada por meio de contagem de células viáveis nos frascos de cultura após 1, 3, 5 e 7 dias do cultivo celular. Foram obtidos 30 fragmentos dentários a partir de dentes extraídos por razões periodontais. Os fragmentos foram raspados com curetas Gracey e condicionados com solução em gel de EDTA a 24% durante 3 minutos, lavados com solução de soro fisiológico, secos e posicionados em placas de 24 poços. Foram incubadas sobre os fragmentos tratados 1x104 células GO por 24 horas, seguido por fixação e preparo para análise por microscopia eletrônica de varredura (MEV). O número de células aderidas sobre os fragmentos foi analisado nas fotomicrografias. O padrão de crescimento das células GO foi compatível com células ósseas, com modificação do padrão do crescimento com o aumento do número de passagens. Houve atividade de fosfatase alcalina em meio osteogênico e convencional, com pico máximo aos 7 dias e atividade de mineralização estimulada ou não por meio osteogênico, com pico máximo aos 21 dias. A análise por meio de citometria de fluxo demonstrou que as células GO não expressaram CD105 e CD166 na 14a passagem, indicando sua diferenciação celular avançada nesse período. A adição de rhPDGF-BB resultou em mudança na taxa de proliferação celular, observando-se pico máximo de crescimento aos 7 dias, com diferenças estatisticamente significantes (p < 0.005; ANOVA post hoc Tukey) em relação aos períodos de 1, 3 e 5 dias. O ensaio de MTT demonstrou maior viabilidade celular no período de 48 hs, comparativamente aos períodos de 24 e 72 horas, quando a densidade óptica celular diminuiu de forma significativa (p< 0.05; Friedmann pósteste Dunn). No ensaio de adesão celular, pode-se observar que a adição de rhPDGFBB aumentou significativamente o número de células aderidas aos fragmentos dentários (p< 0.05; teste t não pareado com correção Welch), com alteração da morfologia celular. Esses resultados sugerem que as células GO tem características compatíveis com linhagem de células osteoblásticas, de fenótipo mais diferenciado após a 12a passagem. A adição de rhPDGF-BB (300ng/ml) resulta em aumento da taxa de proliferação das células GO e do número de células aderidas a fragmentos radiculares, indicando que, nesta concentração, o fator de crescimento é citocompatível, favorecendo a proliferação e adesão celular.(AU)

The goal of this study was to investigate the effects of recombinant human platelet derived growth factor (rhPDGF-BB) at the concentration of 300ng/ml in the proliferation and adhesion of human bone granulation cells to periodontally diseased root fragments. At the first stage of the study, the granulation tissue existent in healing sockets (21 days after its creation) was collected from two systemically healthy nonsmoking adults to the establishment of primary culture. The in vitro properties of bone granulation (BG) cell lineage were characterized by cell viability, MTT, alkaline phosphatase activity and mineralization assays. The effects of culture medium (control) and rhPGDF-BB 300ng/ml (test) in the proliferation and adhesion of BG cells were investigated. The rate of BG cells proliferation was investigated by the number of viable cells present at 1, 3, 5 and 7 days after platting. Thirty root fragments were obtained from teeth extracted for periodontal reasons. Root fragments were scaled and root planed, conditioned with EDTA 24% for 3 minutes, rinsed in saline solution, air-dryed and positioned in 24-well plates. Each fragment was seeded with 104 BG cells, fixated after 24 hours and prepared for analysis in SEM. The number of cells adhered to the fragments was analysed in photomicrographies. BG cells growth pattern was compatible with osteogenic cell lineage, showing modification with the increasing number of cell passage. GO cells expressed alkaline phosphatase activity in conventional and osteogenic culture medium, with maximum peak at 7 days, as well as mineralization activity stimulated or not by osteogenic or non-osteogenic culture medium, with maximum peak at 21 days. The analysis by flow cytometer showed that BG cells have not expressed CD105 and CD106 at the 14th passage, indicating its advanced cell differentiation. The addition of rhPDGF-BB resulted in modification of proliferation rate, with maximum peak observed at 7 days, significantly different from 1-, 3- and 5-day periods (p< 0.005; ANOVA post hoc Tukey). MTT assay showed greater cell viability after 48 hours than after 24 and 72 hours, when optical density has significantly diminished (p< 0.05; Friedmann post hoc Dunn). At cell adhesion assay, it could be observed that the adhesion of rhPDGF-BB has significantly increased the number of cells adhered to root fragments (p< 0.05; unpaired t test with Welchs correction), and alterations in cell morphology. These results suggest that BG cells present in vitro characteristics compatible with osteoblastic cell lineages, with a more differentiated phenotype after the 12th passage. The addition of rhPDGF-BB (300 ng/ml) results in increase of the rate of BG cell proliferation and in the number of cells adhered to root fragments, indicating that, at this concentration, the growth factor is compatible with BG cells and favors cells proliferation and adhesion.(AU)

Reactive Soft Tissue Preservation in Maxillary Large Bone Defects.

PURPOSE: Granulation tissue containing reactive soft tissue with potential multipotent stem cells can help socket healing following extraction. The aim of this study was to assess bone healing of maxillary large bone defects while maintaining reactive soft tissue. MATERIALS AND METHODS: A total of 32 patients presenting large bone defects were selected for this prospective study. Eight patients (Group A) presented with large bone defects but an intact buccal cortical plate, while 24 patients (Group B) presented with large bone defects lacking a buccal cortical plate. Teeth were extracted, and reactive soft tissue was left in the defects. Bone volume was assessed through cone beam computed tomography (CBCT) both before tooth extraction and at 4 months. A histomorphometric evaluation was performed. RESULTS: CBCT and cylinder bone cores were obtained for histology and histomorphometry analysis. At 4 months after tooth extraction, CBCT showed bone volume preservation and bone formation and no statistically significant difference in bone volume before and after tooth extraction in group A. However, in group B, over the same time period, a statistically significant increase (P < .01) of vertical bone volume was reported. Biopsy specimens showed the presence of vital bone in the defects 4 months later. CONCLUSION: Reactive soft tissue left in large bone defects after tooth extraction may support a significant bone volume gain and vital bone formation.

Different configuration of socket shield technique in peri-implant bone preservation: An experimental study in dog mandible.

The aim of this study was to evaluate the influence of the residual root and peri implant bone dimensions on the clinical success of the socket shield technique. Thirty-six dental implants were installed in 6 dogs. The clinical crowns of teeth P3, P4 and M1 were beheaded. Afterwards, the roots were worn down 2-3mm in apical direction until they were located at crestal level. Posterior implant beds were prepared in the center of the roots passing by 3mm apically forming 6 groups in accordance to the remaining root thickness. Radiography of the crestal bone level was performed on day 0 and after 12 weeks. Histomorphometric analyses of the specimens were carried out to measure the crestal bone level, the bone to implant contact and the buccal and lingual bone thickness at the implant shoulder portion. Correlations between groups were analyzed through nonparametric Friedman test, statistical significance was set as p<0.05. All 36 implants were osseointegrated, but 3 samples showed a clinical inflammatory reaction and some radicular fragments presented a small resorption process. On the buccal and lingual side, the radicular fragment was attached to the buccal bone plate by a physiologic periodontal ligament. In the areas where there was space between the implant and the fragment, newly formed bone was demonstrated directly on the implant surface. Within the limitations of an animal pilot study, root-T belt technique may be beneficial in preserving and protecting the bundle bone and preservation of soft tissues. If the thickness of the buccal bone is 3mm, and the thickness of the remaining root fragment is 2mm, the socket shield technique is more predictable and the bone contours can be maintained.

Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket.

Graphene oxide (GO) consisting of a carbon monolayer has been widely investigated for tissue engineering platforms because of its unique properties. For this study, we fabricated a GO-applied scaffold and assessed the cellular and tissue behaviors in the scaffold. A preclinical test was conducted to ascertain whether the GO scaffold promoted bone induction in dog tooth extraction sockets. For this study, GO scaffolds were prepared by coating the surface of a collagen sponge scaffold with 0.1 and 1 µg/mL GO dispersion. Scaffolds were characterized using scanning electron microscopy (SEM), physical testing, cell seeding, and rat subcutaneous implant testing. Then a GO scaffold was implanted into a dog tooth extraction socket. Histological observations were made at 2 weeks postsurgery. SEM observations show that GO attached to the surface of collagen scaffold struts. The GO scaffold exhibited an interconnected structure resembling that of control subjects. GO application improved the physical strength, enzyme resistance, and adsorption of calcium and proteins. Cytocompatibility tests showed that GO application significantly increased osteoblastic MC3T3-E1 cell proliferation. In addition, an assessment of rat subcutaneous tissue response revealed that implantation of 1 µg/mL GO scaffold stimulated cellular ingrowth behavior, suggesting that the GO scaffold exhibited good biocompatibility. The tissue ingrowth area and DNA contents of 1 µg/mL GO scaffold were, respectively, approximately 2.5-fold and 1.4-fold greater than those of the control. Particularly, the infiltration of ED2-positive (M2) macrophages and blood vessels were prominent in the GO scaffold. Dog bone-formation tests showed that 1 µg/mL GO scaffold implantation enhanced bone formation. New bone formation following GO scaffold implantation was enhanced fivefold compared to that in control subjects. These results suggest that GO was biocompatible and had high bone-formation capability for the scaffold. The GO scaffold is expected to be beneficial for bone tissue engineering therapy.

Regional Acceleratory Phenomenon.

The regional acceleratory phenomenon (RAP) is a tissue reaction to a noxious stimulus that increases the healing capacities of the affected tissues. It is typical not only of hard tissues such as bone and cartilage, but also of soft tissues. The RAP is characterized by acceleration of the normal cellular activities, as an 'SOS' phenomenon of the body that has to respond to the new perturbation. In the alveolar bone, the RAP is characterized, at a cellular level, by increased activation of the basic multicellular units (BMUs), thereby increasing the remodeling space. At the tissue level, the RAP is characterized by the production of woven bone, with the typical unorganized pattern, that will be reorganized into lamellar bone at a later stage. In the alveolar bone, the RAP occurs typically in the healing process of the alveolar sockets after tooth extraction, in periodontal disease, after surgery and trauma and during orthodontic tooth movement. In relation to orthodontic tooth movement, the RAP can be seen as a tissue response to the mechanical cyclical perturbation that induces the formation of microdamage that has to be removed to avoid their accumulation and the following bone failure. The adaptation to the new orthodontically induced mechanical environment is ensured by an increased activation of the BMU that returns to normal levels after few months.

Mesenchymal stem/progenitor cell isolation from tooth extraction sockets.

Bone marrow-derived mesenchymal stem/progenitor cells (BMSCs) are commonly used in regeneration therapy. The current primary source of BMSCs is the iliac crest; however, the procedure is associated with various burdens on the patient, including the risk of pain and infection. Hence, the possibility to collect BMSCs from other, more accessible, sources would be an attractive approach. It is well known that stem cells migrate from surrounding tissues and play important roles in wound healing. We thus hypothesized that stem/progenitor cells could be isolated from granulation tissue in the dental socket, and we subsequently collected granulation tissue from dog dental socket 3 d after tooth extraction. After enzyme digestion of the collected tissue, the cells forming colonies constituted the dental socket-derived stem/progenitor cells (dDSCs). Next, dDSCs were compared with dog BMSCs (dBMSCs) for phenotype characterization. A flow cytometric analysis showed that dDSCs were positive for CD44, CD90, and CD271 but negative for CD34 and CD45, similar to dBMSCs. dDSCs also exhibited osteogenic, adipogenic, and chondrogenic differentiation ability, similar to dBMSCs, with a higher capacity for colony formation, proliferation, and motility than dBMSCs. In addition, an in vivo ectopic bone formation assay showed that dDSCs and dBMSCs both induced hard tissue formation, although only dDSCs formed a fibrous tissue-like structure connected to the newly formed bone. Finally, we tested the ability of dDSCs to regenerate periodontal tissue in a one-wall defect model. The defects in the dDSC-transplanted group (ß-TCP/PGA/dDSCs) were regenerated with cementum-like and periodontal ligament-like tissues and alveolar bone, whereas only bony tissue was observed in the control group (ß-TCP/PGA). In conclusion, we identified and characterized a population of stem/progenitor cells in granulation tissue obtained from the dental socket that exhibited several characteristics similar to those of BMSCs. Dental sockets could therefore be a novel source for isolating stem/progenitor cells from bone.

Ultrastructural and immunohistochemical study of early repair of alveolar sockets after the extraction of molars from alendronate-treated rats.

The aim of the present research was to analyze ultrastructural and immunohistochemical aspects of the alveolar repair after the extraction of molars of alendronate (ALN)-treated rats. Wistar rats received 2.5mg/kg body wt/day of ALN during 14 days previously and 7, 14 and 21 days after the extraction of the second mandibular molar. Specimens were fixed in 2% glutaraldehyde + 2.5% formaldehyde under microwave irradiation, decalcified in 4.13% EDTA and paraffin embedded for TRAP histochemistry and immunohistochemistry for OPN, BSP and endoglin, or embedded in Spurr epoxy resin for TEM analysis. Additional specimens had their soft tissues removed and were processed for scanning electron microscopy. The ALN group presented latent TRAP-positive osteoclasts and nonresorbed alveolar crests with bacterial infection. Mild bone necrosis signs were observed at all time points studied. Ultrastructurally, empty osteocyte lacunae were observed and bone trabeculae surface presented hyalinized aspect. A significant delay in alveolar repair occurred, as well as decreased angiogenesis. ALN treatment provoked mild signs of bone necrosis, despite the high dose employed. The present findings add new information about the ultrastructural aspect of the early repair of rats under ALN treatment and highlight for giving attention when oral surgeries are performed in patients using this drug.

Caracterização morfométrica e molecar do papel da iNOS no processo de reparo ósseo alveolar em condições homeostáticas e infecciosas / Morphometric and molecular characterization of the role of iNOS in alveolar bone repair process in homeostatic and infectious conditions

A óxido nítrico sintase induzível (iNOS) é uma enzima responsável pela síntese do óxido nítrico, envolvido na regulação de vários processos fisiológicos, com destaque para relevantes efeitos sobre o tecido ósseo. Entretanto, o papel da iNOS no reparo ósseo alveolar permanece pouco conhecido. Assim, o objetivo deste estudo foi caracterizar o papel da iNOS no processo de reparo ósseo alveolar pósexodontia em condições homeostáticas (controle) e infecciosas em camundongos. Para isso, foram utilizados animais das linhagens WT (C57Bl/6) e iNOSKO, divididos em 2 grupos, condição homeostática (exodontia do incisivo superior direito) e alveolite experimental (induzida pela inoculação de uma suspensão de cultura bacteriana), e analisados de acordo com os diferentes períodos experimentais (0, 7, 14 e 21 dias pós-exodontia), através da análise histomorfométrica e molecular. Na análise histomorfométrica, avaliou-se a densidade de volume das estruturas referentes ao coágulo sanguíneo, tecido conjuntivo e tecido ósseo. Na análise molecular, quantificou-se a expressão de mRNA codificando genes de marcadores do metabolismo ósseo; marcadores de osteoclastos; citocinas e quimiocinas, através de reações de RealTimePCR. A expressão de iNOS esteve presente durante todo o processo de reparo ósseo alveolar nos camundongos C57Bl/6, porém de maneira mais expressiva no período de 7 dias pós-exodontia, e se mostrou aumentada pela indução de alveolite. A análise histomorfométrica demonstrou discretas alterações no processo de reparo ósseo alveolar, na ausência de iNOS, tanto em condições homeostáticas quanto infecciosas. Em condições homeostáticas, a ausência de iNOS não impactou de forma significativa o processo de reparo ósseo alveolar pósexodontia, mas se mostrou associada à modulação de vasos sanguíneos. Já em condições infecciosas, a ausência de iNOS se mostrou associada à modulação de células inflamatórias, osteoblastos e osteoclastos. De forma geral, conclui-se que embora...

The inducible nitric oxide synthase (iNOS) is an enzyme responsible for the synthesis of nitric oxide, a reactive molecule involved in the regulation of several physiological processes, highlighting relevant effects on bone tissue. However, the role of iNOS in alveolar bone repair remains unknown. The aim of this study was to characterize the role of iNOS in alveolar bone healing process after dental extraction in homeostatic and infectious conditions in mice. With this aim, WT (C57Bl/6) and iNOSKO mice strains were divided into 2 groups, homeostatic condition (extraction of the upper right incisor) and experimental alveolitis (induced by inoculation of a suspension of bacterial culture), and analyzed according to the different experimental periods (0, 7, 14 and 21 days post-extraction), through molecular and histomorphometric analysis. In histomorphometric analysis, the volume density of structures related to blood clot, tissue and bone were evaluated. The molecular analysis quantified the expression of mRNA encoding genes of defined as bone metabolism markers; osteoclast markers, as well cytokines and chemokines through RealTimePCR reactions. The expression of iNOS was detected during the all process of the alveolar bone repair in C57Bl/6 mice, with an expression peak at 7 days post-extraction time point, and was significantly enhanced by alveolitis induction. Histomorphometric analysis showed small changes in alveolar bone repair process in the absence of iNOS, in both homeostatic as infectious conditions. Under homeostatic conditions, the absence of iNOS did not impact significantly the process of alveolar bone healing after dental extraction, but it was associated with modulation of blood vessels formation. In the infectious conditions scenario, the absence of iNOS was associated with modulation of inflammatory cells, osteoblasts and osteoclasts counts. In general, it is concluded that even the enzyme iNOS module some aspects of alveolar bone...

Cultura e caracterização de células da granulação óssea in vitro: efeitos proliferativos estimulados por diferentes biomateriais / Culture and characterization of bone granulation cells in vitro: proliferative effects stimulated by different biomaterials

O objetivo deste estudo foi estabelecer cultura primária de células derivadas da granulação óssea (GO) de seres humanos para determinar seu padrão de crescimento in vitro e determinar os efeitos biológicos de três membranas reabsorvíveis feitas de colágeno (BioGide®, GenDerm®, CollaTape®) em culturas de fibroblastos gengivais humanos (FGH) e células da granulação óssea (GO). Foram coletadas amostras de tecido ósseo presente no alvéolo de cicatrização de dois pacientes adultos saudáveis sistemicamente com indicação de cirurgia periodontal regenerativa pela técnica do enxerto ósseo em neoformação. Imediatamente após a coleta, as amostras foram transportadas ao laboratório de cultura de células para estabelecimento da cultura primária. As células foram cultivadas em atmosfera úmida, contendo 5% CO2 a 37oC. A curva de crescimento das células foi determinada por meio de contagem de células viáveis. Após a caracterização da curva de crescimento, foram realizadas a caracterização da amostra por meio de determinação da atividade de fosfatase alcalina e de mineralização. Posteriormente, os efeitos de três diferentes tipos de membranas colágenas sobre a proliferação de células GO e FGH foram investigados por meio do teste MTT. As amostras foram divididas em oito grupos: (1) células FGH em meio DMEM (C-FGH); (2) células FGH em meio DMEM condicionado com membrana GenDerm (GD-FGH); (3) células FGH em meio DMEM condicionado com membrana BioGide (BG-FGH); (4) células FGH em meio DMEM condicionado com membrana ColaTape (CT-FGH); (5) células GO em meio DMEM (C-GO); (6) células GO em meio DMEM condicionado com membrana GenDerm (GD-GO); (7) células GO em meio DMEM condicionado com membrana BioGide (BG-GO); (8) células GO em meio DMEM condicionado com membrana CollaTape (CT-GO). O teste de proliferação celular mostrou que houve aumento significativo (p< 0.05; ANOVA para medidas repetidas) do número de células vitais presentes na cultura nos dias 3 (90,8%)...

The aim of this study was to establish primary culture of cells derived from human bone granulation tissue (GO) in order to determine its growth pattern in vitro and the biological effects of three absorbable collagen membranes (BioGide®, GenDerm®, CollaTape®) in human gingival fibroblasts (FGH) and human bone granulation (GO) cell cultures. Samples of bone tissue present at healing sockets of two systemically healthy adults with indication of periodontal regenerative therapy by the newly forming bone were collected. Immediately after, samples were transported to the laboratory of cell culture to the establishment of primary cultures. Cells were cultivated in humid atmosphere with 95% CO2 at 37oC. Cells growth pattern were determined by counting of viable cells. After characterization of growth pattern, samples were characterized according to alkaline phosphatase activity and mineralization detected by alizarin red. Afterwards, the effects of three different types of collagen membranes on GO and FGH cells were investigated by MTT test. Samples were divided into eight groups: (1) FGH cells in DMEM (C-FGH); (2) FGH in DMEM conditioned by GenDerm® membrane (GD-FGH); (3) FGH in DMEM conditioned with BioGide® (BG-FGH); (4) FGH in DMEM conditioned by CollaTape® (CT-FGH); (5) GO cells in DMEM (C-GO); (6) GO cells in DMEM conditioned by GenDerm® (GD-GO); (7) GO cells in DMEM conditioned by BioGide® (BG-GO); (8) GO cells in DMEM conditioned by CollaTape® (CT-GO). Cell proliferation test showed a significant (p< 0.05; ANOVA for repeated measures) increase in the number of vital cells present in the culture at days 3 (90.8%), 5 (132.50%), 7 (137.50%) and 10 (227.50%) compared to control (dia 0). It was observed alkaline phosphatase activity and mineralization in vitro. There was an increase in the number of FGH and GO viable cells at all groups (p< 0.05; ANOVA for repeated measures). Greater proliferative effect at FGH and GO cells at GD and CT...

Osteogenic potential of effective bone engineering using dental pulp stem cells, bone marrow stem cells, and periosteal cells for osseointegration of dental implants.

PURPOSE: The aim of this comparative study was to investigate cell-based effective bone engineering and the correlation between the osseointegration of dental implants and tissue-engineered bone using dental pulp stem cells (DPSC), bone marrow stem cells (BMSC), and periosteal cells (PC). MATERIALS AND METHODS: The first molar and all premolars were extracted from the mandibles of three dogs, and in each dog, six bone defects (three on each side) were prepared with a 10-mm-diameter trephine bur after 4 weeks. Different materials were implanted in the defects and the sites were allowed to heal. The experimental groups were as follows: (1) dog DPSC and platelet-rich plasma (PRP) (dDPSC/PRP), (2) dog BMSC and PRP (dBMSC/PRP), (3) dog PC and PRP (dPC/PRP), and (4) control (defect only). Eight weeks later, dental implants were placed in the defects. After another 8 weeks, the amount of bone regeneration was assessed by histologic and histomorphometric analyses (bone-implant contact). RESULTS: The mean bone-implant contact values were 66.7% ± 3.6% for group 1 (dDPSC/PRP), 62.5% ± 3.1% for group 2 (dBMSC/PRP), 39.4% ± 2.4% for group 3 (dPC/PRP), and 30.3% ± 2.6% for the control group. CONCLUSIONS: DPSC showed the highest osteogenic potential and may be a useful cell source for tissue-engineered bone around dental implants.

A morphological analysis on the osteocytic lacunar canalicular system in bone surrounding dental implants.

Osseointegration is the most preferable interface of dental implants and newly formed bone. However, the cavity preparation for dental implants often gives rise to empty lacunae or pyknotic osteocytes in bone surrounding the dental implant. This study aimed to examine the chronological alternation of osteocytes in the bone surrounding the titanium implants using a rat model. The distribution of the osteocytic lacunar canalicular system (OLCS) in bone around the titanium implants was examined by silver impregnation according to Bodian's staining. We also performed double staining for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), as well as immunohistochemistry for fibroblast growth factor (FGF) 23--a regulator for the serum concentration of phosphorus--and sclerostin, which has been shown to inhibit osteoblastic activities. Newly formed bone and the injured bone at the early stage exhibited an irregularly distributed OLCS and a few osteocytes positive for sclerostin or FGF23, therefore indicating immature bone. Osteocytes in the surrounding bone from Day 20 to Month 2 came to reveal an intense immunoreactivity for sclerostin. Later on, the physiological bone remodeling gradually replaced such immature bone into a compact profile bearing a regularly arranged OLCS. As the bone was remodeled, FGF23 immunoreactivity became more intense, but sclerostin became less so in the well-arranged OLCS. In summary, it seems likely that OLCS in the bone surrounding the dental implants is damaged by cavity formation, but later gradually recovers as bone remodeling takes place, ultimately inducing mature bone.

Response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate membrane.

This study investigated the response of human alveolar bone-derived cells to a novel poly(vinylidene fluoride-trifluoroethylene)/barium titanate (P(VDF-TrFE)/BT) membrane. Osteoblastic cells were cultured in osteogenic conditions either on P(VDF-TrFE)/BT or polytetrafluoroethylene (PTFE) for up to 14 days. At 7 and 14 days, the mRNA expression of Runt-related transcription factor 2 (RUNX2), Type I collagen (COL I), Osteopontin (OPN), Alkaline phosphatase (ALP), Bone sialoprotein (BSP), and Osteocalcin (OC), key markers of the osteoblastic phenotype, and of Bcl2-associated X protein (Bax), B-cell CLL/lymphoma 2 (Bcl-2), and Survivin (SUR), associated with the control of the apoptotic cell death, was assayed by real-time PCR. In situ ALP activity was qualitatively evaluated by means of Fast red staining. Surface characterization was also qualitatively and quantitatively assayed in terms of topography, roughness, and wettability. Cells grown on P(VDF-TrFE)/BT exhibited a significantly higher mRNA expression for all markers compared to the ones on PTFE, except for Bcl-2, which was not detected for both groups. Additionally, Fast red staining was noticeably stronger in cultures on P(VDF-TrFE)/BT at 7 and 14 days. At micron- and submicron scale, SEM images and roughness analysis revealed that PTFE and P(VDF-TrFE)/BT exhibited a smooth topography and a similar roughness, respectively. PTFE membrane displayed higher contact angles compared with P(VDF-TrFE)/BT, as indicated by wettability assay. The novel P(VDF-TrFE)/BT membrane supports the acquisition of the osteoblastic phenotype in vitro, while up-regulating the expression of apoptotic markers. Further in vivo experiments should be carried out to confirm the capacity of P(VDF-TrFE)/BT membrane in promoting bone formation in guided bone regeneration.

Dexamethasone effects on Na(v)1.6 in tooth pulp, dental nerves, and alveolar osteoclasts of adult rats.

Dexamethasone causes extensive physiologic reactions including the reduction of inflammation and pain. Here, we asked whether it also affected dental or periodontal cells or dental innervation by altering voltage-gated sodium channel Na(v)1.6 immunoreactivity (IR) or neural synaptophysin. Daily dexamethasone (0.2 mg/kg) given for 1 week to rats caused 12-fold increased intensity of Na(v)1.6-IR in dendritic pulpal cells of normal molars and incisors compared with vehicle treatment. These cells also co-localized monocyte (ED-1) or dendritic cell (CD11b/Ox42) markers, and their location in molars expanded during dexamethasone treatment to include deeper pulp. Furthermore, dexamethasone caused a 10-fold decrease in the number of Na(v)1.6-immunoreactive multinucleate osteoclasts along the alveolar bone of molar root sockets. No changes occurred for neural Na(v)1.6 at axonal nodes of Ranvier, even though IR for calcitonin gene-related peptide was greatly decreased, as expected, and neural synaptophysin-IR was decreased 59% by dexamethasone. At 4 days after tooth injury, pulpal vasodilation and increased Na(v)1.6-immunoreactive pulp cells were similar for all groups. Thus, dexamethasone changes dental pulp cell and alveolar osteoclast Na(v)1.6-IR in normal teeth, but different mechanisms occur after tooth injury when tissue reactions were similar for dexamethasone- and vehicle-treated rats. Steroid-induced alterations of dental pain and inflammation coincide with altered exocytic capability in dental nerve fibers as shown by synaptophysin-IR and with altered pulp cell Na(v)1.6-IR and osteoclast number, but not with any changes in Na(v)1.6-IR for nodes of Ranvier in myelinated dental axons.

Immunolocalization of CSF-1, RANKL and OPG in the enamel-related periodontium of the rat incisor and their implications for alveolar bone remodeling.

The enamel-related periodontium (ERP) in rat incisors is related to bone resorption. In these teeth the face of the socket related to the enamel is continuously removed at the inner side and newly formed at the outer side. CSF-1, RANKL and OPG are regulatory molecules essential for osteoclastogenesis. To verify the effects of impeded eruption on bone remodeling, the tooth eruption was prevented by immobilization of lower rat incisor and CSF-1, RANKL and OPG distribution in the ERP was analyzed after 18 days of immobilization and in normal eruption. The region of the alveolar crest of the rat incisor was used. Immunohistochemistry and tartrate-resistant acid phosphatase (TRAP) were performed. The immunostaining of the dental follicle was quantified using Leica QWin software. Positive-TRAP osteoclasts were counted, and both groups were compared. In the normal incisor, the number of osteoclasts was significantly greater than in the immobilized tooth. In the dental follicle, there was no significant difference in the immunostaining intensity for CSF-1 and OPG between the groups (p > 0.05), but for RANKL the immobilized incisor group showed immunostaining intensity smaller than the normal incisor group (p < 0.01). These findings suggest that changes in the ERP, in the immobilized incisor, modify the RANKL/OPG ratio, in the presence of CSF-1, altering the metabolism of cells that participate in the bone remodeling.

Scintigraphic evaluation of early osteoblastic activity in extraction sockets treated with platelet-rich plasma.

PURPOSE: The aim of this study was to investigate the early effect of platelet-rich plasma (PRP) on osteoblastic activity during the healing process of soft tissue impacted mandibular third molar extraction sockets by means of bone scintigraphy. PATIENTS AND METHODS: Twelve patients with bilaterally soft tissue impacted mandibular third molars were included in the study. The impacted right and left mandibular third molars were surgically extracted in the same session. PRP was administered randomly into the extraction sockets in the study (S) group whereas the extraction sockets in the control (C) group were left without PRP treatment. Scintigrams were obtained in the first and fourth weeks after surgery to evaluate the osteoblastic activity within extraction sockets in both groups. RESULTS: Scintigraphic findings of postoperative first and fourth weeks did not show significantly increased osteoblastic activity between S group and C group (P > .05). However, the osteoblastic activity in both groups significantly increased in postoperative week 4 in comparison to week 1 (P < .05). CONCLUSION: The application of PRP alone into soft tissue impacted mandibular third molar extraction sockets failed to increase the osteoblastic activity in postsurgical weeks 1 and 4 in comparison to non-PRP-treated sockets.

Osteogenic potential of embryonic stem cells in tooth sockets.

Embryonic stem cells (ESCs) are established from blastocysts and give rise to various types of cells and tissues. In the present study, we assessed the osteogenic potential of ESCs using in vitro culture conditions and in vivo differentiation in tooth sockets. An ESC-derived embryoid body (EB) was formed and subsequently induced to an osteogenic lineage. The differentiated EB cells exhibited increased expression of various osteogenic markers as determined by real-time PCR analysis. Likewise, the differentiated EB-derived cells had enhanced alkaline phosphatase activity and calcium accumulation, as determined by cytochemical methods. For in vivo transplantation, mixtures of ESCs and hydroxyapatite/ tricalcium phosphate particles or EBs alone were transplanted into female rat tooth sockets. After 12 weeks, we observed formation of osteogenic structure in the tooth sockets without evidence of teratomas. These data suggest that pluripotent ESCs can serve as an alternative source for the reconstruction of craniofacial structures, as well as for further applications.

[Human osteoblasts attachment to guided tissue regeneration membranes which were coated either with platelet-rich plasma or platelet-poor plasma].

OBJECTIVE: To determine whether human alveolar bone osteoblasts (HABO) attachment to commercial available guided tissue regeneration (GTR) membranes can be enhanced by coating with freshly prepared human platelet-rich plasma (PRP) and platelet poor plasma (PPP). METHODS: Human osteoblasts established from tissue explants were used at 4 th passage in culture. Human whole blood from healthy subjects was collected and centrifuged twice to produce the PRP fraction and PPP fraction. Double-sided adhesive tape was used to fix 3 mm discs of each membrane and cover-slides to the bottom of a 24-well tissue culture plate. A (GoreTex-ePTFE), B (GoreTex-Resolut) and C (Inion-GTR) membranes were studied. Cover-slides were positive control. Membranes or cover-slides were exposed to PRP, PPP or PBS respectively for 2 hours. Membranes and cover-slides were seeded with osteoblasts (5 x 10(7) cells/L) and allowed to attach for 24 hours. After staining with hematoxylin, the number of attached cells per mm(2) was counted using a light microscope with graticule. The the ultrastructure of osteoblasts attachment to the membranes was observed by scanning electronic microscopy. RESULTS: PRP and PPP-treated membranes significantly enhanced osteoblasts attachment compared to PBS-treated membranes (P < 0.05). There was more osteoblasts attachment in the PRP-treated membranes than in the PPP-treated membranes (P < 0.05). Cover-slides showed more osteoblasts attachment than the three membranes (P < 0.05). B and C membranes showed higher cell attachment than A membranes (P < 0.05). SEM showed that osteoblasts attached to the membranes treated by PRP were spindle and stretched well, and there were platelets, fibrins in a interlaced mesh on the membranes, which appeared to grow in a multiplayer style. The osteoblasts attached to the membranes treated by PPP or PBS were round and partially attached. CONCLUSIONS: PRP and PPP could improve attachment of osteoblasts in the three membranes, and PRP altered and enhanced the way of the attachment to the membranes.