Mesenchymal stem cells

Cell based therapies as they apply to tissue engineering and regenerative medicine, require cells capable of self renewal and differentiation, and a prerequisite is to be able to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies therefore figures as an integral part of tissue engineering. Stem cells serve as a reserve for biological repair, having the potential to differentiate into a number of specialised cell types within the body; they therefore represent the most useful candidates for cell based therapies. The primary goal of stem cell research is to produce cells that are both patient specific, as well as having properties suitable for the specific conditions for which they are intended to remedy. From a purely scientific perspective, stem cells allow scientists to gain a deeper understanding of developmental biology and regenerative therapies. Stem cells have acquired a number of uses for applications in regenerative medicine, immunotherapy, gene therapy, but it is in the area of tissue engineering that they generate most excitement, primarily as a result of their capacity for self-renewal and pluripotency. A unique feature of stem cells is their ability to maintain an uncommitted quiescent state in vivo and then, once triggered by conditions such as disease, injury or natural wear or tear, serve as a reservoir and natural support system to replenish lost cells. Although these cells retain the plasticity to differentiate into various tissues, being able to control this differentiation process is still one of the biggest challenges facing stem cell research. In an effort to harness the potential of these cells a number of studies have been conducted using both embryonic/foetal and adult stem cells. The use of embryonic stem cells (ESC) have been hampered by strong ethical and political concerns, this despite their perceived versatility due to their pluripotency. Ethical issues aside, other concerns raised with ESCs relates to the possibility of tumorigenesis, immune rejection and complications with immunosuppressive therapies, all of which adds layers of complications to the application ESC in research and which has led to the search for alternative sources for stem cells. The adult tissues in higher organisms harbours cells, termed adult stem cells, and these cells are reminiscent of unprogrammed stem cells. A number of sources of adult stem cells have been described. Bone marrow is by far the most accessible source of two potent populations of adult stem cells, namely haematopoietic stem cells (HSCs) and bone marrow mesenchymal stem cells (BMSCs). Autologously harvested adult stem cells can, in contrast to embryonic stem cells, readily be used in autografts, since immune rejection is not an issue; and their use in scientific research has not attracted the ethical concerns which have been the case with embryonic stem cells. The major limitation to their use, however, is the fact that adult stem cells are exceedingly rare in most tissues. This fact makes identifying and isolating these cells problematic; bone marrow being perhaps the only notable exception. Unlike the case of HSCs, there are as yet no rigorous criteria for characterizing MSCs. Changing acuity about the pluripotency of MSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to MSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their study in vitro. Also, when MSCs are cultured in vitro, there is a loss of the in vivo microenvironment, resulting in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage numbers in culture, characterized by the onset of senescence related changes. As a consequence, it is necessary to establish protocols for generating large numbers of MSCs but without affecting their differentiation potential. MSCs are capable of differentiating into mesenchymal tissue lineages, including bone, cartilage, fat, tendon, muscle, and marrow stroma. Recent findings indicate that adult bone marrow may also contain cells that can differentiate into the mature, nonhematopoietic cells of a number of tissues, including cells of the liver, kidney, lung, skin, gastrointestinal tract, and myocytes of heart and skeletal muscle. MSCs can readily be expanded in vitro and can be genetically modified by viral vectors and be induced to differentiate into specific cell lineages by changing the microenvironment–properties which makes these cells ideal vehicles for cellular gene therapy. MSCs can also exert profound immunosuppressive effects via modulation of both cellular and innate immune pathways, and this property allows them to overcome the issue of immune rejection. Despite the many attractive features associated with MSCs, there are still many hurdles to overcome before these cells are readily available for use in clinical applications. The main concern relates to in vivo characterization and identification of MSCs. The lack of a universal biomarker, sparse in vivo distribution, and a steady age related decline in their numbers, makes it an obvious need to decipher the reprogramming pathways and critical molecular players which govern the characteristics unique to MSCs. This book presents a comprehensive insight into the biology of adult stem cells and their utility in current regeneration therapies. The adult stem cell populations reviewed in this book include bone marrow derived MSCs, adipose derived stem cells (ASCs), umbilical cord blood stem cells, and placental stem cells. The features such as MSC circulation and trafficking, neuroprotective properties, and the nurturing roles and differentiation potential of multiple lineages have been discussed in details. In terms of therapeutic applications, the strengths of MSCs have been presented and their roles in disease treatments such as osteoarthritis, Huntington’s disease, periodontal regeneration, and pancreatic islet transplantation have been discussed. An analysis comparing osteoblast differentiation of umbilical cord blood stem cells and MSCs has been reviewed, as has a comparison of human placental stem cells and ASCs, in terms of isolation, identification and therapeutic applications of ASC in bone, cartilage regeneration, as well as myocardial regeneration. It is my sincere hope that this book will update the reader as to the research progress of MSC biology and potential use of these cells in clinical applications. It will be the best reward to all contributors of this book, if their efforts herein may in some way help the readers in any part of their study, research, and career development.

Identification of bone morphogenetic proteins 2 and 4 in commercial demineralized freeze-dried bone allograft preparations : pilot study

BACKGROUND: Demineralized freeze-dried bone allografts (DFDBAs) have been proposed as a useful adjunct in periodontal therapy to induce periodontal regeneration through the induction of new bone formation. The presence of bone morphogenetic proteins (BMPs) within the demineralized matrix has been proposed as a possible mechanism through which DFDBA may exert its biologic effect. However, in recent years, the predictability of results using DFDBA has been variable and has led to its use being questioned. One reason for the variability in tissue response may be attributed to differences in the processing of DFDBA, which may lead to loss of activity of any bioactive substances within the DFDBA matrix. Therefore, the purpose of this investigation was to determine whether there are detectable levels of bone morphogenetic proteins in commercial DFDBA preparations. METHODS: A single preparation of DFDBA was obtained from three commercial sources. Each preparation was studied in triplicate. Proteins within the DFDBA samples were first extracted with 4M guanidinium HCI for seven days at 40 degrees celsius and the residue was further extracted with 4M guanidinium HCL/EDTA for seven days at 40 degrees celsius. Two anti-human BMP-2 and -4 antibodies were used for the detection of the presence of BMP's in the extracts. RESULTS: Neither BMP-2 nor BMP-4 was detected in any of the extracts. When recombinant human BMP-2 and -4 were added throughout the extraction process of DFDBA extraction, not only were intact proteins detected but smaller molecular weight fragments were also noted in the extract. CONCLUSIONS: These results indicate that all of the DFDBA samples tested had no detectable amounts of BMP-2 and -4. In addition, an unknown substance present in the DFDBA may be responsible for degradation of whatever BMPs might be present.

Lithium release from ß-tricalcium phosphate inducing cementogenic and osteogenic differentiation for both hPDLCs and hBMSCs

It is accepted that the accelerated differentiation of tissue cells on bioactive materials is of great importance to regenerate the lost tissues. It was previously reported that lithium (Li) ions could enhance the in vitro proliferation and differentiation of retinoblastoma cells and endometrium epithelia by activating the Wnt canonical signalling pathway. It is interesting to incorporate Li ions into bioactive ceramics, such as β-tricalcium phosphate (Li-β-TCP), in order to stimulate both osteogenic and cementogenic differentiation of different stem cells for the regeneration of bone/periodontal tissues. Therefore, the aim of this study was to investigate the interactions of human periodontal ligament cells (hPDLCs) and human bone marrow stromal cells (hBMSCs) with Li-β-TCP bioceramic bulks and their ionic extracts, and further explore the osteogenic and cementogenic stimulation of Li-β-TCP bioceramics and the possible molecular mechanisms. The results showed that Li-β-TCP bioceramic disks supported the cell attachment and proliferation, and significantly enhanced bone/cementum-related gene expression, Wnt canonical signalling pathway activation for both hPDLCs and hBMSCs, compared to conventional β-TCP bioceramic disks without Li. The release of Li from Li-β-TCP powders could significantly promote the bone/cementum-related gene expression for both hPDLCs and hBMSCs compared to pure β-TCP extracts without Li release. Our results suggest that the combination of Li with β-TCP bioceramics may be a promising method to enhance bone/cementum regeneration as Li-β-TCP possesses excellent in vitro osteogenic and cementogenic stimulation properties by inducing bone/cementum-related gene expression in both hPDLCs and hBMSCs.

Associations among Emdogain®, human oral carcinoma cells and oral proteolytic enzymes

The Enamel matrix derivative Emdogain® (EMD) is a commercially available tissue extract preparation of porcine enamel origin. Studies have shown EMD to be clinically useful in promoting periodontal regeneration. EMD has been widely used in periodontal therapy for over ten years, but the mechanism of its action and the exact composition are not completely clear. EMD is predominantly amelogenin (>90%). However, unlike amelogenin, EMD has a number of growth factor-like effects and it has been shown to enhance the proliferation, migration and other cellular functions of periodontal ligament fibroblasts and osteoblasts. In contrast, the effects of EMD on epithelial cell lines and in particular on oral malignant cells have not been adequately studied. In addition, EMD has effects on the production of cytokines by several oral cell lines and the product is in constant interaction with different oral enzymes. Regardless of the various unknown properties of EMD, it is said to be clinically safe in regenerative procedures, also in medically compromised patients. The aim of the study was to examine whether gingival crevicular fluid (GCF), which contains several different proteolysis enzymes, could degrade EMD and alter its biological functions. In addition, the objective was to study the effects of EMD on carcinogenesis-related factors, in particular MMPs, using in vitro and in vivo models. This study also aimed to contribute to the understanding of the composition of EMD. GCF was capable of degrading EMD, depending on the periodontal status, with markedly more degradation in all states of periodontal disease compared to healthy controls. EMD was observed to stimulate the migration of periodontal ligament fibroblasts (PLF), whereas EMD together with GCF could not stimulate this proliferation. In addition, recombinant amelogenin, the main component of EMD, decreased the migration of PLFs. A comparison of changes induced by EMD and TGF-β1 in the gene profiles of carcinoma cells showed TGF-β1 to regulate a greater number of genes than EMD. However, both of the study reagents enhanced the expression of MMP-10 and MMP-9. Furthermore, EMD was found to induce several factors closely related to carcinogenesis on gene, protein, cell and in vivo levels. EMD enhanced the production of MMP-2, MMP-9 and MMP-10 proteins by cultured carcinoma cells. In addition, EMD stimulated the migration and in vitro wound closure of carcinoma cells. EMD was also capable of promoting metastasis formation in mice. In conclusion, the diseased GCF, containing various proteases, causes degradation of EMD and decreased proliferation of PLFs. Thus, this in vitro study suggests that the regenerative effect of EMD may decrease due to proteases present in periodontal tissues during the inflammation and healing of the tissues in vivo. Furthermore, EMD was observed to enhance several carcinoma-related factors and in particular the production of MMPs by benign and malignant cell lines. These findings suggest that the clinical safety of EMD with regard to dysplastic mucosal lesions should be further investigated.

Uso de biomateriais nas abordagens cirúrgicas minimamente invasivas para tratamento de defeitos ósseos periodontais: revisão sistemática

O objetivo da presente dissertação foi realizar uma revisão sistemática para investigar os efeitos do uso de biomateriais no tratamento de defeitos ósseos periodontais por meio de abordagens cirúrgicas minimamente invasivas sobre os parâmetros clínicos periodontais. Foi realizada uma busca por ensaios randomizados controlados publicados entre os anos de 1995 e 2013 nas bases MEDLINE, EMBASE, CENTRAL, BVS, CLINICALTRIALS.gov, abstracts da International Association for Dental Research (IADR) e busca manual por possíveis estudos a serem incluídos. Foram localizados 541 estudos, sendo 12 selecionados para avaliação da elegibilidade e 5 estudos incluídos para análise qualitativa. Após aplicação de um instrumento para análise do viés associado, demonstrou-se um baixo risco de viés associado à maioria dos estudos incluídos. Nos resultados dos estudos incluídos foi possível observar que embora bem-sucedida, a associação de diferentes biomateriais às técnicas minimamente invasivas não trouxe benefício adicional aos parâmetros clínicos avaliados.

Influence of enamel matrix derivative (Emdogain((R))) and sodium fluoride on the healing process in delayed tooth replantation: histologic and histometric analysis in rats

Although it has already been shown that enamel matrix derivative (Emdogain((R))) promotes periodontal regeneration in the treatment of intrabony periodontal defects, there is little information concerning its regenerative capacity in cases of delayed tooth replantation. To evaluate the alterations in the periodontal healing of replanted teeth after use of Emdogain((R)), the central incisors of 24 Wistar rats (Rattus norvegicus albinus) were extracted and left on the bench for 6 h. Thereafter, the dental papilla and the enamel organ of each tooth were sectioned for pulp removal by a retrograde way and the canal was irrigated with 1% sodium hypochlorite. The teeth were assigned to two groups:in group I, root surface was treated with 1% sodium hypochlorite for 10 min (changing the solution every 5 min), rinsed with saline for 10 min and immersed in 2% acidulated-phosphate sodium fluoride for 10 min; in group II, root surfaces were treated in the same way as described above, except for the application of Emdogain((R)) instead of sodium fluoride. The teeth were filled with calcium hydroxide (in group II right before Emdogain((R)) was applied) and replanted. All animals received antibiotic therapy. The rats were killed by anesthetic overdose 10 and 60 days after replantation. The pieces containing the replanted teeth were removed, fixated, decalcified and paraffin-embedded. Semi-serial 6-mu m-thick sections were obtained and stained with hematoxylin and eosin for histologic and histometric analyses. The use of 2% acidulated-phosphate sodium fluoride provided more areas of replacement resorption. The use of Emdogain((R)) resulted in more areas of ankylosis and was therefore not able to avoid dentoalveolar ankylosis. It may be concluded that neither 2% acidulated-phosphate sodium fluoride nor Emdogain((R)) were able to prevent root resorption in delayed tooth replantation in rats.

Orthodontic movement of teeth with intraosseous defects: histologic and histometric study in dogs

The purpose of this study was to evaluate histologically, in dogs, the periodontal healing of 1-walled intraosseous defects in teeth that were subjected to orthodontic movement toward the defects. The defects were surgically created bilaterally at the mesial aspects of the maxillary second premolars and distal aspects of the mandibular second premolars of 4 mongrel dogs. One week after creating the defects, an orthodontic appliance was installed, and the teeth were randomly assigned to 1 of 2 treatment groups: those in the test group received a titanium-molybdenum alloy rectangular wire spring that performed a controlled tipping root movement, and those in the control group received a passive stainless steel wire. Active orthodontic movement of the test teeth lasted 2 months and was followed by a stabilization period of another 2 months, after which the animals were killed. Throughout the study, routine daily plaque control was performed on the dogs with a topical application of a 2% chlorhexicline gel. The results showed no difference between the groups, with some regularization of the defects and periodontal regeneration limited to the apical portion of the defects. Histometric analysis showed a significant difference in bone height; on average, it was 0.53 mm smaller in the test group. It was concluded that orthodontic movement does not interfere with the healing of 1-walled intraosseous defects, with the exception of the linear extent of new bone apposition.

Platelet-rich plasma stimulates cytokine expression and alkaline phosphatase activity in osteoblast-derived osteosarcoma cells

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

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.

Healing of dehiscence defects following root surface demineralization with tetracycline: A histologic study in monkeys

Background: the purpose of this study was to histologically evaluate the healing of experimental dehiscence defects after surface demineralization with tetracycline hydrochloride.Methods: Six adult male monkeys (Cebus apella) were used in this study. Dehiscence defects were surgically created on the buccal aspect of the mandibular lateral incisors in all animals. The root surfaces were debrided and planed. In a split-mouth design, a 10% tetracycline hydrochloride solution was applied to one tooth for 4 minutes (T group), followed by irrigation with saline. The contralateral tooth served as a control (C group). The flaps were repositioned and sutured. The animals were sacrificed at 6 months postoperatively and histological sections were processed. Computer-assisted histomorphometric analysis was used to evaluate the formation of new cementum, new bone, new connective tissue attachment, and length of the epithelium (junctional and sulcular).Results: Bone regeneration was similar in both groups (1.5 +/- 0.3 mm for the T group and 1.5 +/- 0.6 mm for the C group). The C group showed more new cementum than the T group (2.3 +/- 0.3 mm versus 2.2 +/- 0.3 mm) as well as a longer epithelium (1.0 +/- 0.3 mm versus 0.9 +/- 0.2 mm). The T group presented more new connective tissue attachment (3.1 +/- 0.2 mm) than the C group (2.9 +/- 0.6 mm). However, no statistically significant differences were detected between the two groups.Conclusions: the amount of new attachment was similar in both groups. Root conditioning with 10% tetracycline solution did not produce any additional new attachment in comparison to the controls.

Autogenous bone graft with or without a calcium sulfate barrier in the treatment of class II furcation defects: A histologic and histometric study in dogs

Background: The purpose of this study was to histologically evaluate the healing of surgically created Class II furcation defects treated using an autogenous bone (AB) graft with or without a calcium sulfate (CS) barrier. Methods: The second, third, and fourth mandibular premolars (P2, P3, and P4) of six mongrel dogs were used in this study. Class II furcation defects (5 mm in height × 2 mm in depth) were surgically created and immediately treated. Teeth were randomly divided into three groups: group C (control), in which the defect was filled with blood clot; group AB, in which the defect was filled with AB graft; and group AB/CS, in which the defect was filled with AB graft and covered by a CS barrier. Elaps were repositioned to cover all defects. The animals were euthanized 90 days post-surgery. Mesio-distal serial sections were obtained and stained with either hematoxylin and eosin or Masson's trichrome. Histometric, using image-analysis software, and histologic analyses were performed. Linear and area measurements of periodontal healing were evaluated and calculated as a percentage of the original defect. Percentage data were transformed into arccosine for statistical analysis (analysis of variance; P<0.05). Results: Periodontal regeneration in the three groups was similar. Regeneration of bone and connective tissue in the furcation defects was incomplete in most of the specimens. Statistically significant differences were not found in any of the evaluated parameters among the groups. Conclusion: Periodontal healing was similar using surgical debridement alone, AB graft, or AB graft with a CS barrier in the treatment of Class II furcation defects.

Blood cells attachment after root conditioning and PRP application: An in vitro study

Aim: Root conditioning is aimed at smear layer removal and at dental matrix collagen exposure, which may promote periodontal regeneration. This in vitro study assessed smear layer removal, collagen fiber exposure and the influence of PRP (platelet-rich plasma) application on adhesion of blood cells to the root surface using scanning electron microscopy (SEM). Materials and methods: Scaled root samples (n = 160) were set in five groups and conditioned with: group I - control group (saline solution); group II (EDTA 24%); group III (citric acid 25%); group IV (tetracycline hydrochloride 50 mg/ml); group V (sodium citrate 30%). Eighty samples were assessed using the root surface modification index (RSMI). The other eighty samples were set in two groups. The first group (n = 40) received PRP gel application with a soft brush and the second group (n = 40) received PRP application and then a blood drop. The fibrin clot formation was assessed in the first group and the blood cells adhesion was assessed in the second group using the BEAI (blood elements adhesion index). A previously trained, calibrated, and blind examiner evaluated photomicrographs. Statistical analysis was performed using the Kruskal-Wallis's and Dunn's tests. Results: Group III attained the best results for RSMI and BEAI. Moreover, it was the only group showing fibrin clot formation. Conclusion: Citric acid was the most efficient conditioner for smear layer removal, collagen fiber exposure and blood cell adhesion. Moreover, it was the only group showing fibrin clot formation after PRP application. Clinical significance: This study demonstrated that root conditioning followed by PRP application may favor blood cell adhesion on root surface which may optimize periodontal healing.

Enxerto de osso autógeno e barreira de sulfato de cálcio no tratamento de defeitos de furca classe II. Estudo histológico e histométrico em cães

Pós-graduação em Odontologia - FOA

Efeito da movimentação ortodôntica sobre lesão de furca grau II tratada com matriz mineral óssea bovina e membrana absorvível: estudo histológico e histométrico em cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Proteína derivada da matriz do esmalte associada à regeneração tecidual guiada e vidro bioativo no tratamento de lesões de furca grau III. Estudo histológico em cães

Pós-graduação em Odontologia - FOAR