Highly efficient neural differentiation of human somatic stem cells, isolated by minimally invasive periodontal surgery

Neural stem cells (NSCs) are potential sources for cell therapy of neurodegenerative diseases and for drug screening. Despite their potential benefits, ethical and practical considerations limit the application of NSCs derived from human embryonic stem cells (ES) or adult brain tissue. Thus, alternative sources are required to satisfy the criteria of ready accessibility, rapid expansion in chemically defined media and reliable induction to a neuronal fate. We isolated somatic stem cells from the human periodontium that were collected during minimally invasive periodontal access flap surgery as part of guided tissue regeneration therapy. These cells could be propagated as neurospheres in serum-free medium, which underscores their cranial neural crest cell origin. Culture in the presence of epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) under serum-free conditions resulted in large numbers of nestin-positive/Sox-2-positive NSCs. These periodontium-derived (pd) NSCs are highly proliferative and migrate in response to chemokines that have been described as inducing NSC migration. We used immunocytochemical techniques and RT-PCR analysis to assess neural differentiation after treatment of the expanded cells with a novel induction medium. Adherence to substrate, growth factor deprivation, and retinoic acid treatment led to the acquisition of neuronal morphology and stable expression of markers of neuronal differentiation by more than 90% of the cells. Thus, our novel method might provide nearly limitless numbers of neuronal precursors from a readily accessible autologous adult human source, which could be used as a platform for further experimental studies and has potential therapeutic implications.

Collagen membranes at immediate implants: a histomorphometric study in dogs

Aim To evaluate the influence of resorbable membranes on hard tissue alterations and osseointegration at implants placed into extraction sockets in a dog model. Material and methods In the mandibular premolar region, implants were installed immediately into the extraction sockets of six Labrador dogs. Collagen-resorbable membranes were placed at the test sites, while the control sites were left uncovered. Implants were intended to heal in a submerged mode. After 4 months of healing, the animals were sacrificed, and ground sections were obtained for histomorphometric evaluation. Results After 4 months of healing, a control implant was not integrated (n=5). Both at the test and at the control sites, bone resorption occurred. While the most coronal bone-to-implant contact was similar between the test and the control sites, the alveolar bone crest outline was maintained to a higher degree at the buccal aspect of the test sites (loss: 1.7 mm) compared with the control sites (loss: 2.2 mm). Conclusions The use of collagen-resorbable membranes at implants immediately placed into extraction sockets contributed to a partial (23%) preservation of the buccal outline of the alveolar process. To cite this article:Caneva M, Botticelli D, Salata LA, Souza SLS, Carvalho Cardoso L, Lang NP. Collagen membranes at immediate implants: a histomorphometric study in dogs.Clin. Oral Impl. Res. 21, 2010; 891-897.doi: 10.1111/j.1600-0501.2010.01946.x.

The Bone Formation Capabilities of the Anorganic Bone Matrix-Synthetic Cell-Binding Peptide 15 Grafts in an Animal Periodontal Model: A Histologic and Histomorphometric Study in Dogs

Background: The aim of this study is to verify the regenerative potential of particulate anorganic bone matrix synthetic peptide-15 (ABM-P-15) in class III furcation defects associated or not with expanded polytetrafluoroethylene membranes. Methods: Class III furcation defects were produced in the mandibular premolars (P2, P3, and P4) of six dogs and filled with impression material. The membranes and the bone grafts were inserted into P3 and P4, which were randomized to form the test and control groups, respectively; P2 was the negative control group. The animals were sacrificed 3 months post-treatment. Results: Histologically, the complete closure of class III furcation defects was not observed in any of the groups. Partial periodontal regeneration with similar morphologic characteristics among the groups was observed, however, through the formation of new cementum, periodontal ligament, and bone above the notch. Histologic analysis showed granules from the bone graft surrounded by immature bone matrix and encircled by newly formed tissue in the test group. The new bone formation area found in the negative control group was 2.28 +/- 2.49 mm(2) and in the test group it was 6.52 +/- 5.69 mm(2), which showed statistically significant differences for these groups considering this parameter (Friedman test P <0.05). There was no statistically significant difference among the negative control, control, and test groups for the other parameters. Conclusions: The regenerative potential of ABM-P-15 was demonstrated through new bone formation circumscribing and above the graft particles. The new bone also was accompanied by the formation of new cementum and periodontal ligament fibers. J Periodontol 2010;81:594-603.

Influence of implant positioning in extraction sockets on osseointegration: histomorphometric analyses in dogs

Aim To evaluate the influence of implant positioning into extraction sockets on osseointegration. Material and methods Implants were installed immediately into extraction sockets in the mandibles of six Labrador dogs. In the control sites, the implants were positioned in the center of the alveolus, while in the test sites, the implants were positioned 0.8 mm deeper and more lingually. After 4 months of healing, the resorptive patterns of the alveolar crest were evaluated histomorphometrically. Results All implants were integrated in mineralized bone, mainly composed of mature lamellar bone. The alveolar crest underwent resorption at the control as well as at the test sites. After 4 months of healing, at the buccal aspects of the control and test sites, the location of the implant rough/smooth limit to the alveolar crest was 2 +/- 0.9 mm and 0.6 +/- 0.9 mm, respectively (P < 0.05). At the lingual aspect, the bony crest was located 0.4 mm apically and 0.2 mm coronally to the implant rough/smooth limit at the control and test sites, respectively (NS). Conclusions From a clinical point of view, implants installed into extraction sockets should be positioned approximately 1 mm deeper than the level of the buccal alveolar crest and in a lingual position in relation to the center of the alveolus in order to reduce or eliminate the exposure above the alveolar crest of the endosseous (rough) portion of the implant. To cite this article:Caneva M, Salata LA, de Souza SS, Baffone G, Lang NP, Botticelli D. Influence of implant positioning in extraction sockets on osseointegration: histomorphometric analyses in dogs.Clin. Oral Impl. Res. 21, 2010; 43-49.

Histological Outcomes on the Development of New Space-making Devices for Maxillary Sinus Floor Augmentation

Background: Previous studies have pointed out that the mere elevation of the maxillary sinus membrane promotes bone formation without the use of augmentation materials. Purpose: This experimental study aimed at evaluating if the two-stage procedure for sinus floor augmentation could benefit from the use of a space-making device in order to increase the bone volume to enable later implant installation with good primary stability. Materials and Methods: Six male tufted capuchin primates (Cebus apella) were subjected to extraction of the three premolars and the first molar on both sides of the maxilla to create an edentulous area. The sinuses were opened using the lateral bone-wall window technique, and the membrane was elevated. One resorbable space-making device was inserted in each maxillary sinus, and the bone window was returned in place. The animals were euthanatized after 6 months, and biopsy blocks containing the whole maxillary sinus and surrounding soft tissues were prepared for ground sections. Results: The histological examination of the specimens showed bone formation in contact with both the schneiderian membrane and the device in most cases even when the device was displaced. The process of bone formation indicates that this technique is potentially useful for two-stage sinus floor augmentation. The lack of stabilization of the device within the sinus demands further improvement of space-makers for predictable bone augmentation. Conclusions: It is concluded that (1) the device used in this study did not trigger any important inflammatory reaction; (2) when the sinus membrane was elevated, bone formation was a constant finding; and (3) an ideal space-making device should be stable and elevate the membrane to ensure a maintained connection between the membrane and the secluded space.

Ridge Preservation With Acellular Dermal Matrix and Anorganic Bone Matrix Cell-Binding Peptide P-15 After Tooth Extraction in Humans

Background: Preventing ridge collapse with the extraction of maxillary anterior teeth is vital to an esthetic restorative result. Several regenerative techniques are available and are used for socket preservation. The aim of this study is to analyze by clinical parameters the use of acellular dermal matrix (ADM) and anorganic bovine bone matrix (ABM) with synthetic cell-binding peptide P-15 to preserve alveolar bone after tooth extraction. Methods: Eighteen patients in need of extraction of maxillary anterior teeth were selected and randomly assigned to the test group (ADM plus ABM/P-15) or the control group (ADM only). Clinical measurements were recorded initially and at 6 months after ridge-preservation procedures. Results: In the clinical measurements (external vertical palatal measurement [EVPM], external vertical buccal measurement [EVBM], and alveolar horizontal measurement [AHM]) the statistical analysis showed no difference between test and control groups initially and at 6 months. The intragroup analysis, after 6 months, showed a statistically significant reduction in the measurements for both groups. In the comparison between the two groups, the differences in the test group were as follows: EVPM = 0.83 +/- 1.53 mm; EVBM = 1.20 +/- 2.02 mm; and AHM = 2.53 +/- 1.81 mm. The differences in the control group were as follows: EVPM = 0.87 +/- 1.13 mm; EVBM = 1.50 +/- 1.15 mm; and AHM = 3.40 +/- 1.39 mm. The differences in EVPM and EVBM were not statistically significant; however, in horizontal measurement (AHM), there was a statistically significant difference (P<0.05). Conclusion: The results of this study show that ADM used as membrane associated with ABM/P-15 can be used to reduce buccal-palatal dimensions compared to ADM alone for preservation of the alveolar ridge after extraction of anterior maxillary teeth. J Periodontol 2011;82:72-79.

Functionalization of dendrimers for improved gene delivery to mesenchymal stem cell

Disease, injury, and age problems compromise human quality of life and continuously motivate the search for new and more efficacious therapeutic approaches. The field of Tissue Regeneration and Engineering has greatly evolved over the last years, mainly due to the combination of the important advances verified in Biomaterials Science and Engineering with those of Cell and Molecular Biology. In particular, a new and promising area arose – Nanomedicine – that takes advantage of the extremely small size and especial chemical and physical properties of Nanomaterials, offering powerful tools for health improvement. Research on Stem Cells, the self-renewing progenitors of body tissues, is also challenging to the medical and scientific communities, being expectable the appearance of new and exciting stem cell-based therapies in the next years. The control of cell behavior (namely, of cell proliferation and differentiation) is of key importance in devising strategies for Tissue Regeneration and Engineering. Cytokines, growth factors, transcription factors and other signaling molecules, most of them proteins, have been identified and found to regulate and support tissue development and regeneration. However, the application of these molecules in long-term regenerative processes requires their continuous presence at high concentrations as they usually present short half-lives at physiological conditions and may be rapidly cleared from the body. Alternatively, genes encoding such proteins can be introduced inside cells and be expressed using cell’s machinery, allowing an extended and more sustained production of the protein of interest (gene therapy). Genetic engineering of stem cells is particularly attractive because of their self-renewal capability and differentiation potential. For Tissue Regeneration and Engineering purposes, the patient’s own stem cells can be genetically engineered in vitro and, after, introduced in the body (with or without a scaffold) where they will not only modulate the behavior of native cells (stem cell-mediated gene therapy), but also directly participate in tissue repair. Cells can be genetically engineered using viral and non-viral systems. Viruses, as a result of millions of years of evolution, are very effective for the delivery of genes in several types of cells, including cells from primary sources. However, the risks associated with their use (like infection and immunogenic reactions) are driving the search for non-viral systems that will efficiently deliver genetic material into cells. Among them, chemical methods that are promising and being investigated use cationic molecules as carriers for DNA. In this case, gene delivery and gene expression level remain relatively low when primary cells are used. The main goal of this thesis was to develop and assess the in vitro potential of polyamidoamine (PAMAM) dendrimers based carriers to deliver genes to mesenchymal stem cells (MSCs). PAMAM dendrimers are monodispersive, hyperbranched and nanospherical molecules presenting unique characteristics that make them very attractive vehicles for both drug and gene delivery. Although they have been explored for gene delivery in a wide range of cell lines, the interaction and the usefulness of these molecules in the delivery of genes to MSCs remains a field to be explored. Adult MSCs were chosen for the studies due to their potential biomedical applications (they are considered multipotent cells) and because they present several advantages over embryonic stem cells, such as easy accessibility and the inexistence of ethical restrictions to their use. This thesis is divided in 5 interconnected chapters. Chapter I provides an overview of the current literature concerning the various non-viral systems investigated for gene delivery in MSCs. Attention is devoted to physical methods, as well as to chemical methods that make use of polymers (natural and synthetic), liposomes, and inorganic nanoparticles as gene delivery vectors. Also, it summarizes the current applications of genetically engineered mesenchymal stem cells using non-viral systems in regenerative medicine, with special focus on bone tissue regeneration. In Chapter II, the potential of native PAMAM dendrimers with amine termini to transfect MSCs is evaluated. The level of transfection achieved with the dendrimers is, in a first step, studied using a plasmid DNA (pDNA) encoding for the β-galactosidase reporter gene. The effect of dendrimer’s generation, cell passage number, and N:P ratio (where N= number of primary amines in the dendrimer; P= number of phosphate groups in the pDNA backbone) on the level of transfection is evaluated, being the values always very low. In a second step, a pDNA encoding for bone morphogenetic protein-2, a protein that is known for its role in MSCs proliferation and differentiation, is used. The BMP-2 content produced by transfected cells is evaluated by an ELISA assay and its effect on the osteogenic markers is analyzed through several classical assays including alkaline phosphatase activity (an early marker of osteogenesis), osteocalcin production, calcium deposition and mineralized nodules formation (late osteogenesis markers). Results show that a low transfection level is enough to induce in vitro osteogenic differentiation in MSCs. Next, from Chapter III to Chapter V, studies are shown where several strategies are adopted to change the interaction of PAMAM dendrimers with MSCs cell membrane and, as a consequence, to enhance the levels of gene delivery. In Chapter III, generations 5 and 6 of PAMAM dendrimers are surface functionalized with arginine-glycine-aspartic acid (RGD) containing peptides – experiments with dendrimers conjugated to 4, 8 and 16 RGD units were performed. The underlying concept is that by including the RGD integrin-binding motif in the design of the vectors and by forming RGD clusters, the level of transfection will increase as MSCs highly express integrins at their surface. Results show that cellular uptake of functionalized dendrimers and gene expression is enhanced in comparison with the native dendrimers. Furthermore, gene expression is dependent on both the electrostatic interaction established between the dendrimer moiety and the cell surface and the nanocluster RGD density. In Chapter IV, a new family of gene delivery vectors is synthesized consisting of a PAMAM dendrimer (generation 5) core randomly linked at the periphery to alkyl hydrophobic chains that vary in length and number. Herein, the idea is to take advantage of both the cationic nature of the dendrimer and the capacity of lipids to interact with biological membranes. These new vectors show a remarkable capacity for internalizing pDNA, being this effect positively correlated with the –CH2– content present in the hydrophobic corona. Gene expression is also greatly enhanced using the new vectors but, in this case, the higher efficiency is shown by the vectors containing the smallest hydrophobic chains. Finally, chapter V reports the synthesis, characterization and evaluation of novel gene delivery vectors based on PAMAM dendrimers (generation 5) conjugated to peptides with high affinity for MSCs membrane binding - for comparison, experiments are also done with a peptide with low affinity binding properties. These systems present low cytotoxicity and transfection efficiencies superior to those of native dendrimers and partially degraded dendrimers (Superfect®, a commercial product). Furthermore, with this biomimetic approach, the process of gene delivery is shown to be cell surface receptor-mediated. Overall, results show the potential of PAMAM dendrimers to be used, as such or modified, in Tissue Regeneration and Engineering. To our knowledge, this is the first time that PAMAM dendrimers are studied as gene delivery vehicles in this context and using, as target, a cell type with clinical relevancy. It is shown that the cationic nature of PAMAM dendrimers with amine termini can be synergistically combined with surface engineering approaches, which will ultimately result in suitable interactions with the cytoplasmic membrane and enhanced pDNA cellular entry and gene expression. Nevertheless, the quantity of pDNA detected inside cell nucleus is always very small when compared with the bigger amount reaching cytoplasm (accumulation of pDNA is evident in the perinuclear region), suggesting that the main barrier to transfection is the nuclear membrane. Future work can then be envisaged based on the versatility of these systems as biomedical molecular materials, such as the conjugation of PAMAM dendrimers to molecules able to bind nuclear membrane receptors and to promote nuclear translocation.

Influência da laserterapia na proliferação de células-tronco do ligamento periodontal humano

Low level laser irradiation (LLLI) has been used in Dentistry to promote wound healing and tissue regeneration. The literature shows a positive effect of LLLI on cell proliferation, but little is known about their effectiveness in promoting stem cells proliferation. The aim of this study was to evaluate the effect of LLLI on the proliferative rate of human periodontal ligament stem cells. Extracts of periodontal ligament were isolated from two third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were grown in α-MEM culture medium supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were irradiated with a InGaAlP-diode laser, using two different energy densities (0,5J/cm 2 - 16 seconds and 1,0J/cm² - 33 seconds), with wavelength of 660nm and output power of 30mW. A new irradiation, using the same parameters, was performed 48h after the first. A control group (non irradiated) was kept under the same experimental culture conditions. The Trypan blue exclusion test and the mitochondrial activity of the cells measured by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] essay were performed to assess the cell proliferation in the intervals of 0, 24, 48 e 72 h after irradiation. The data of cell counts were submitted to nonparametrical statistical tests (Kruskal-Wallis and Mann-Whitney), considering a confidence interval of 95%. DAPI (4 -6-Diamidino-2-phenylindole) staining of the cells was performed at 72h interval to evaluate possible nuclear morphological changes induced by LLLI. The results of this study show that the energy density of 1,0 J/cm² promoted greater cell proliferation compared to the other groups (control and 0,5 J/cm²) at intervals of 48 and 72h. The mitochondrial activity measured by MTT essay showed similar results to the Trypan blue cell counting test. The group irradiated with 1,0J/cm² exhibited a significantly higher MTT activity in the intervals of 48 and 72h, when compared to the group irradiated with 0,5J/cm². No nuclear morphological change was observed in the cells from the three groups studied. It is concluded that LLLI has stimulatory effects on the proliferation of human periodontal ligament stem cells. Therefore, the use of laser irradiation in this cell type may be important to promote future advances in periodontal regeneration

Uso de membrana biossintética a base de celulose na regeneração tecidual guiada

Guided Tissue Regeneration (GTR) is a regenerative treatment modality that requires the placement of a physical barrier over a bone defect in such a way that the proliferation of the surrounding soft tissues into the barrier-protected area is prevented. Thereby in the guided osseous regeneration allowing bone-forming cells from the existent bone edges to invade the space and produce bone. The physical barriers should be biocompatible, allow cellular occlusion, maintain adequate space, tissue integration and facility in the application. They can be occlusive or permeable, absorbable or non-absorbable. Among various types of physical barriers in the market, the cellulose biosynthetic membrane is emphasizing by the necessary characteristics for GTR and to be a national product developed in low cost.

Análises histológica e morfométrica do uso de membrana biossintética de celulose em trocleoplastia experimental de cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Biomechanical and histologic analysis in aortic endoprosthesis using fibrin glue

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Bone healing in critical-size defects treated with either bone graft, membrane, or a combination of both materials: a histological and histometric study in rat tibiae

Objectives: The aim of the present investigation was to histologically analyze the effect of using lyophilized bovine bone (GenOx (R) organic matrix) with (or without) guided tissue regeneration (using a decalcified cortical osseous membrane [GenDerm (R)]) on bone healing in surgically created critical-size defects created in rat tibia.Material and methods: Surgical critical-size bone defects were created in 64 animals that were randomly divided into four groups: group I (control); group II (defect filled with GenOx (R)); group III (defect covered by GenDerm (R)); group IV (defect filled with GenOx (R) and covered by GenDerm (R)). Animals were killed at 30 or 90 days post-surgery. The specimens were embedded in paraffin, serially cut, and stained with hematoxylin and eosin for analysis under light microscopy. The formation of new bone in the cortical area of the defect was histomorphometrically evaluated.Results: All experimental groups demonstrated superior bone healing compared with the control group. However, group IV samples showed evidence of more advanced healing at both 30 and 90 days post-surgery as compared with the other experimental groups.Conclusions: The bovine organic bone graft GenOx (R) associated with GenDerm (R) this produced the best treatment results in the case of critical-size defects in rat tibia.

Hypoxia Enhances the Angiogenic Potential of Human Dental Pulp Cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Intrusion of teeth with class III furcation: a clinical, histologic and histometric study in dogs

Aim: To assess orthodontic intrusion effects on periodontal tissues in dogs' pre-molars with class III furcations treated with open flap debridement (OFD) or with guided tissue regeneration (GTR) associated to bone autograft (BA).Material and Methods: Class III furcations were created in the pre-molars of seven mongrel dogs. After 75 days, teeth were randomly treated with OFD or GTR/BA. After 1 month, metallic crowns were assembled on pre-molars and connected apically to mini-implants by nickel-titanium springs. Teeth were randomly assigned to orthodontic intrusion (OFD+I and GTR/BA+I) groups or no movement (OFD and GTR/BA) groups. Dogs were sacrificed after 3 months of movement and 1 month retention.Results: All class III furcations were closed or reduced to class II or I in the intrusion groups, while 50% of the lesions in non-moved teeth remained unchanged. Intruded teeth presented higher probing depth and lower gingival marginal level than non-moved teeth (p < 0.01). Clinical attachment gain was reduced in the intrusion groups by the end of retention (p < 0.05). OFD+I presented smaller soft tissue area and larger bone tissue area than other groups (p < 0.05).Conclusion: Orthodontic intrusion with anchorage via mini-implants improved the healing of class III furcation defects after OFD in dogs. GTR/BA impaired those results.

PDGF-B gene therapy accelerates bone engineering and oral implant osseointegration

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 x 10(8) or 5.5 x 10(9) pfu ml (1)), Ad encoding luciferase (Ad-Luc; 5.5 x 10(9) pfu ml (1); control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml (1)). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, microcomputed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo. Gene Therapy (2010) 17, 95-104; doi: 10.1038/gt.2009.117; published online 10 September 2009