Mesenchymal stem cells surpass the capacity of bone marrow aspirate concentrate for periodontal regeneration

Abstract Regenerative approaches using mesenchymal stem cells (MSCs) have been evaluated to promote the complete formation of all missing periodontal tissues, e.g., new cementum, bone, and functional periodontal ligaments. MSCs derived from bone marrow have been applied to bone and periodontal defects in several forms, including bone marrow aspirate concentrate (BMAC) and cultured and isolated bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate the periodontal regeneration capacity of BMAC and cultured BM-MSCs in the wound healing of fenestration defects in rats. Methodology: BM-MSCs were obtained after bone marrow aspiration of the isogenic iliac crests of rats, followed by cultivation and isolation. Autogenous BMAC was collected and centrifuged immediately before surgery. In 36 rats, fenestration defects were created and treated with suspended BM-MSCs, BMAC or left to spontaneously heal (control) (N=6). Their regenerative potential was assessed by microcomputed tomography (µCT) and histomorphometry, as well as their cell phenotype and functionality by the Luminex assay at 15 and 30 postoperative days. Results: BMAC achieved higher bone volume in 30 days than spontaneous healing (p<0.0001) by enhancing osteoblastic lineage commitment maturation, with higher levels of osteopontin (p=0.0013). Defects filled with cultured BM-MSCs achieved higher mature bone formation in early stages than spontaneous healing and BMAC (p=0.0241 and p=0.0143, respectively). Moreover, significantly more cementum-like tissue formation (p<0.0001) was observed with new insertion of fibers in specimens treated with BM-MSCs within 30 days. Conclusion: Both forms of cell transport, BMAC and BM-MSCs, promoted bone formation. However, early bone formation and maturation were achieved when cultured BM-MSCs were used. Likewise, only cultured BM-MSCs were capable of achieving complete periodontal regeneration with inserted fibers in the new cementum-like tissue.

Comparative proteomic analysis of dental cementum from deciduous and permanent teeth.

BACKGROUND AND OBJECTIVES: Dental cementum (DC) is a mineralized tissue covering tooth roots that plays a critical role in dental attachment. Differences in deciduous vs. permanent tooth DC have not been explored. We hypothesized that proteomic analysis of DC matrix would identify compositional differences in deciduous (DecDC) vs. permanent (PermDC) cementum that might reflect physiological or pathological differences, such as root resorption that is physiological in deciduous teeth but can be pathological in the permanent dentition. METHODS: Protein extracts from deciduous (n = 25) and permanent (n = 12) teeth were pooled (five pools of DecDC, five teeth each; four pools of PermDC, three teeth each). Samples were denatured, and proteins were extracted, reduced, alkylated, digested, and analyzed by liquid chromatography-mass spectrometry (LC-MS/MS). The beta-binomial statistical test was applied to normalized spectrum counts with 5% significance level to determine differentially expressed proteins. Immunohistochemistry was used to validate selected proteins. RESULTS: A total of 510 proteins were identified: 123 (24.1%) exclusive to DecDC; 128 (25.1%) exclusive to PermDC; 259 (50.8%) commonly expressed in both DecDC and PermDC. Out of 60 differentially expressed proteins, 17 (28.3%) were detected in DecDC, including myeloperoxidase (MPO), whereas 43 (71.7%) were detected in PermDC, including decorin (DCN) and osteocalcin (BGLAP). Overall, Gene Ontology (GO) analysis indicated that all expressed proteins were related to GO biological processes that included localization and response to stress, and the GO molecular function of differentially expressed proteins was enriched in cell adhesion, molecular binding, cytoskeletal protein binding, structural molecular activity, and macromolecular complex binding. Immunohistochemistry confirmed the trends for selected differentially expressed proteins in human teeth. CONCLUSIONS: Clear differences were found between the proteomes of DecDC and PermDC. These findings may lead to new insights into developmental differences between DecDC and PermDC, as well as to a better understanding of physiological/pathological events such as root resorption.

Cellular therapy in periodontal regeneration.

Periodontitis is a chronic inflammatory condition leading to destruction of the tooth supporting tissues, which if left untreated may cause tooth loss. The treatment of periodontitis mainly aims to arrest the inflammatory process by infection control measures, although in some specific lesions a limited periodontal regeneration can also be attained. Current regenerative approaches are aimed to guide the cells with regenerative capacity to repopulate the lesion and promote new cementum and new connective tissue attachment. The first phase in periodontal tissue regeneration involves the differentiation of mesenchymal cells into cementoblasts to promote new cementum, thus facilitating the attachment of new periodontal ligament fibers to the root and the alveolar bone. Current regenerative approaches limit themselves to the confines of the lesion by promoting the self-regenerative potential of periodontal tissues. With the advent of bioengineered therapies, several studies have investigated the potential use of cell therapies, mainly the use of undifferentiated mesenchymal cells combined with different scaffolds. The understanding of the origin and differentiation patterns of these cells is, therefore, important to elucidate their potential therapeutic use and their comparative efficacy with current technologies. This paper aims to review the in vitro and experimental studies using cell therapies based on application of cementoblasts and mesenchymal stem cells isolated from oral tissues when combined with different scaffolds.

On the discovery of cementum.

Though cementum of the tooth root is critical for periodontal structure and tooth attachment and function, this tissue was not discovered and characterized on human teeth until a full century later than enamel and dentin. Early observations from the seventeenth to the nineteenth centuries by Marcello Malpighi, Antonie van Leeuwenhoek, Robert Blake, Jacques Tenon and Georges Cuvier founded a confusing and conflicting nomenclature that obscured the nature of cementum, often conflating it with bone. Advances in microscopy and histological procedures yielded the first detailed descriptions of human cementum in the 1830s by Jan Purkinje and Anders Retzius, who identified for the first time acellular and cellular types of cementum, and the resident cementocytes embedded in the latter. Comparative anatomy studies by Richard Owen and others over the latter half of the nineteenth century identified coronal and radicular cementum varieties across the Reptilia and Mammalia. The functional importance of cementum was not appreciated until detailed anatomical studies of the periodontium were performed by G.V. Black and others in the late nineteenth and early twentieth centuries. These early studies on cementum laid the foundation for more advanced understanding of cementum ultrastructure, composition, development, physiology, disease, genetics, repair and regeneration throughout the twentieth and into the twenty-first century.

Evaluation of Enterococcus faecalis adhesion, penetration, and method to prevent the penetration of Enterococcus faecalis into root cementum: Confocal laser scanning microscope and scanning electron microscope analysis.

AIM: To ascertain the role of Enterococcus faecalis in persistent infection and a possible method to prevent the penetration of E. faecalis into root cementum. METHODOLOGY: One hundred and twenty human single-rooted extracted teeth divided into five groups. Group I (control): intact teeth, Group II: no apical treatment done, Group III divided into two subgroups. In Groups IIIa and IIIb, root apex treated with lactic acid of acidic and neutral pH, respectively. Group IV: apical root cementum exposed to lactic acid and roughened to mimic the apical resorption. Group V: apical treatment done same as Group IV and root-end filling done using mineral trioxide aggregate (MTA). Apical one-third of all samples immersed in E. faecalis broth for 8 weeks followed by bone morphogenetic protein and obturation and again immersed into broth for 8 weeks. Teeth split into two halves and observed under confocal laser scanning microscope and scanning electron microscope, organism identified by culture and polymerase chain reaction techniques. RESULTS: Adhesion and penetration was observed in Group IIIa and Group IV. Only adhesion in Group II and IIIB and no adhesion and penetration in Group I and V. CONCLUSION: Adhesion and penetration of E. faecalis into root cementum providing a long-term nidus for subsequent infection are the possible reason for persistent infection and root-end filling with MTA prevents the adhesion and penetration.

High-level expression and characterization of a glycosylated human cementum protein 1 with lectin activity.

This work aims to contribute to the knowledge of human cementum protein 1 (CEMP1), its conformational characteristics and influence during the biomineralization process. The results revealed that hrCEMP1 expressed in Pichia pastoris is a 2.4% glycosylated, thermostable protein which possesses a molecular mass of 28,770 Da. The circular dichroism spectrum indicated a secondary structure content of 28.6% of alpha-helix, 9.9% of beta-sheet and 61.5% of random-coil forms. Biological activity assays demonstrated that hrCEMP1 nucleates and regulates hydroxyapatite crystal growth. Hereby, it is demonstrated for the first time that CEMP1 has a (C-type) lectin-like activity and specifically recognizes mannopyranoside. The information produced by this biochemical and structural characterization may contribute to understand more fully the biological functions of CEMP1.

Evaluation of the presence of Enterococcus Faecalis in root cementum: A confocal laser scanning microscope analysis.

AIM: The aim of this study is to address the cause of persistent infection of root cementum by Enterococcus faecalis. MATERIALS AND METHODS: A sample of 60 human single-rooted teeth were divided into three groups. Group I (control group) had no access opening and one-third of the apical root cementum was sealed using varnish. Group II had no preparation of teeth samples. In group III, apical root cementum was exposed to organic acid and roughened using diamond point to mimic apical resorption. After access opening in groups II and III, all teeth samples were sterilized using gamma irradiation (25 kGy). E. faecalis broth was placed in the root canal and apical one-third of the tooth was immersed in the broth for 8 weeks with alternate day refreshment followed by biomechanical preparation, obturation and coronal seal. Apical one-third of all teeth samples were again immersed in the broth for 8 weeks with alternate day refreshment to mimic secondary infection. The samples were observed under a confocal microscope after splitting the teeth into two halves. RESULTS: E. faecalis penetrated 160 µm deep into the root cementum in group III samples and only showed adhesion in group II samples. CONCLUSION: Penetration and survival of E. faecalis deep inside the cementum in extreme conditions could be the reason for persistent infection.