A comparative in vitro and in vivo study of porcine- and bovine-derived non-cross-linked collagen membranes.

The porcine-derived non-cross-linked collagen membrane Bio-gide® (BG) and the bovine-derived non-cross-linked collagen membrane Heal-all® (HA) were compared to better understand their in vitro biophysical characteristics and in vivo degradation patterns as a reference for clinical applications. It was showed that the porosity, specific surface area, pore volume and pore diameter of BG were larger than those of HA (64.5 ± 5.2% vs. 48.6 ± 6.1%; 18.6 ± 2.8 m2 /g vs. 2.3 ± 0.6 m2 /g; 0.114 ± 0.002 cm3 /g vs. 0.003 ± 0.001 cm3 /g; 24.4 ± 3.5 nm vs. 7.3 ± 1.7 nm, respectively); the average swelling ratio of BG was higher than that of HA (412.6 ± 41.2% vs. 270.0 ± 2.7%); the tensile strength of both dry and wet HA was higher than those of BG (18.26 ± 3.27 MPa vs. 4.02 ± 1.35 MPa; 2.24 ± 0.21 MPa vs. 0.16 ± 0.02 MPa, respectively); 73% of HA remained after 72 h in collagenase solution, whereas only 8.2% of BG remained. A subcutaneous rat implantation model revealed that, at 3, 7, 14, 28, and 56 days postmembrane implantation, there were more total inflammatory cells, especially more M1 and M2 polarized macrophages and higher M2/M1 ratio in BG than in HA; in addition, the fibrous capsule around BG was also thicker than that around HA. Moreover, concentrations of dozens of cytokines including interleukin-2(IL-2), IL-7, IL-10 and so forth. in BG were higher than those in HA. It is suggested that BG and HA might be suitable for different clinical applications according to their different characteristics.

A Modified Minimally Invasive Approach for the Treatment of Mandibular Condylar Fracture.

ABSTRACT: In this study, we introduced a small "V" shaped incision around the ear lobe modified from fact-lift approach to treat the mandibular condylar neck and subcondylar fractures to further minimize and hide the postsurgical scars. A total of 15 cases with mandibular condylar fractures from low levels of condylar neck to high levels of subcondylar region were treated by this approach. An incision was designed from the posterior rim of the tragus down to the anterior skin wrinkle to the ear lobe, surrounding it, continually up and backward for a little distance, forming a "V"-like shape. After cutting and elevation of the skin flap, the remaining steps were the same as those in trans-parotid mini-invasive approaches. The surgical scars introduced in this study were more hidden than those in other mini-invasive approaches and all patients were satisfied with their aesthetic appearances. We suggested that the modified "V" shaped mini-invasive approach has an advantage in achieving aesthetic results and could be an alternative incision in treating some types of mandibular condylar and subcondylar fractures.

Multinucleated Giant Cells Induced by a Silk Fibroin Construct Express Proinflammatory Agents: An Immunohistological Study.

Multinucleated giant cells (MNGCs) are frequently observed in the implantation areas of different biomaterials. The main aim of the present study was to analyze the long-term polarization pattern of the pro- and anti-inflammatory phenotypes of macrophages and MNGCs for 180 days to better understand their role in the success or failure of biomaterials. For this purpose, silk fibroin (SF) was implanted in a subcutaneous implantation model of Wistar rats as a model for biomaterial-induced MNGCs. A sham operation was used as a control for physiological wound healing. The expression of different inflammatory markers (proinflammatory M1: CCR-7, iNos; anti-inflammatory M2: CD-206, CD-163) and tartrate-resistant acid phosphatase (TRAP) and CD-68 were identified using immunohistochemical staining. The results showed significantly higher numbers of macrophages and MNGCs within the implantation bed of SF-expressed M1 markers, compared to M2 markers. Interestingly, the expression of proinflammatory markers was sustained over the long observation period of 180 days. By contrast, the control group showed a peak of M1 macrophages only on day 3. Thereafter, the inflammatory pattern shifted to M2 macrophages. No MNGCs were observed in the control group. To the best of our knowledge, this is study is the first to outline the persistence of pro-inflammatory MNGCs within the implantation bed of SF and to describe their long-term kinetics over 180 days. Clinically, these results are highly relevant to understand the role of biomaterial-induced MNGCs in the long term. These findings suggest that tailored physicochemical properties may be a key to avoiding extensive inflammatory reactions and achieving clinical success. Therefore, further research is needed to elucidate the correlation between proinflammatory MNGCs and the physicochemical characteristics of the implanted biomaterial.

Evaluation of Osteogenic/Cementogenic Modulating Potential of PAI-1 Transfected Media for Stem Cells.

AIM OF THE STUDY: In vitro evaluation of the effects of plasminogen activator inhibitor-1 (PAI-1) transfected-conditioned media (P-CM) on the differentiation of human periodontal ligament stem cells (hPDLSCs) and human periapical follicular stem cells (hPAFSCs). MATERIALS AND METHODS: The hPDLSCs and hPAFSCs received from impacted third molars were treated with P-CM and viability, as well as differentiation of the cells were evaluated. Plasmids were constructed according to standard techniques, and all sequences were validated by proper enzyme digestion and sequencing. Chinese hamster ovarian (CHO) cells were transfected with pcDNA3.1-hPAI-1 plasmid to obtain P-CM, followed by western blotting and PAI-1-specific ELISA kit to evaluate the proteins of P-CM. The cell viability of hPDLSCs and hPAFSCs were analyzed using MTT assay after 48 h of incubation. Alizarin red S staining was performed to evaluate the differentiation of hPDLSCs and hPAFSCs. The reverse transcription-polymerase chain reaction was used to observe the expression levels of osteogenic/cementogenic marker genes. The human cytokine antibody array was applied for further analysis of cytokine expression in P-CM. RESULTS: P-CM significantly promoted the differentiation of hPDLSCs and hPAFSCs and upregulated the expression of osteogenic/cementogenic marker genes in vitro. Furthermore, rhPAI-1 promoted mineralized nodules formation of hPDLSCs and hPAFSCs, and we identified that other proteins, RANTES and IL-6, were highly expressed in P-CM. CONCLUSIONS: P-CM promoted the differentiation of hPDLSCs and hPAFSCs by upregulating the expression of RANTES and IL-6, and interaction between PAI-1 and RANTES/IL-6 signaling may be involved in P-CM-induced osteogenic/cementogenic differentiation.

Biomaterial-induced multinucleated giant cells express proinflammatory signaling molecules: A histological study in humans.

The biomaterials physicochemical characteristics influence their cellular reaction, degradation and regenerative capacities. Macrophages and multinucleated giant cells (MNGCs) are observed in the augmentation area of biomaterials. This study, for the first time, evaluated the polarization pattern of macrophages and MNGCs in response to two different bone substitute materials (synthetic bone substitute material [SBSM] = NanoBone vs. xenogeneic bone substitute material [XBSM] = Bio-Oss) in human bone biopsies compared to non-augmented bone (control). Histomorphometrical analysis of the polarization in proinflammatory (M1) and anti-inflammatory (M2) cells was performed using different immunohistochemical markers: CD-68 = macrophages; CCR-7 and Cox-2 (M1) and CD-206 and CD-163 (M2) and tartrate-resistant acid phosphatase (TRAP). The macrophage polarization pattern in SBSM showed a significantly higher number of M1 cells than did XBSM and non-augmented bone. XBSM induced a significantly higher number of CD-206-positive macrophages than SBSM did. No significant difference was found between XBSM and the non-augmented bone. MNGCs expressed CD-68 and TRAP. In both test-groups, MNGCs showed a high proinflammatory character (CCR-7 and Cox-2-positive) and their number in the SBSM group was significantly higher than that of XBSM. The tissue distribution showed a significantly low percentage of the remaining biomaterial in SBSM compared to XBSM. Within the limitations of this study, these findings show that MNGCs exhibit a rather proinflammatory character and lead to biomaterial degradation, once they are induced in a high number. The premature degradation of bone substitute materials is compensated with a high percentage of connective tissue and not new bone formation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 780-790, 2019.

A high current anodization to fabricate a nano-porous structure on the surface of Ti-based implants.

In this study, an oxide layer on Ti-based implants is fabricated by using a high current anodization (HCA) technique in the nitrate electrolyte. This layer is composed of micro-pits and nano-porous arrays in the honeycomb structure. The results show that both the roughness and the layer thickness are related to the reaction time, whereas the size of nano-pores has little to do with the anodization duration. Compared to the nano-tubular arrays constructed by the conventional anodization, this nano-porous layer shows significantly improved mechanical stability. Furthermore, the in vitro assay of osteoblasts shows that cells behaviors on this surface can be modulated by the topology of this special layer. A suitable hierarchical structure composed of micro-pits and nano-porous structure can significantly stimulate osteoblasts attachment, activity, spreading and ALP function. Therefore, this hierarchical surface layer may provide a promising approach, which endows the Ti-based implants with better stability and osseointegration.

Sugar-based collagen membrane cross-linking increases barrier capacity of membranes.

OBJECTIVES: This study examines the permeability and barrier capacity of a sugar cross-linked resorbable collagen membrane ex vivo and in vivo. MATERIALS AND METHODS: In an ex vivo study, injectable platelet-rich fibrin (i-PRF), a peripheral blood-derived human leukocyte-and-platelet-rich plasma was used to analyze membrane permeability. in vivo subcutaneous implantation in Wistar rats (n = 4 per time point and group) was used to investigate the barrier capacity of the membrane. The induced in vivo cellular reaction was evaluated at 3, 15, and 30 days and compared to sham OP (control) without biomaterial using histological, immunohistochemical, and histomorphometric methods. RESULTS: Ex vivo, the membrane was impenetrable to leukocytes, platelets, and fibrin from peripheral human blood concentrate (PRF). In vivo, the membrane maintained its structure and remained impervious to cells, connective tissue, and vessels over 30 days. CD-68-positive cell (macrophage) numbers significantly decreased from 3 to 15 days, while from day 15 onwards, the number of multinucleated giant cells (MNGCs) increased significantly. Correspondingly, a rise in implantation bed vascularization from 15 to 30 days was observed. However, no signs of degradation or material breakdown were observed at any time point. CONCLUSION: Ex vivo and in vivo results showed material impermeability to cellular infiltration of human and murine cells, which highlights the membrane capacity to serve as a barrier over 30 days. However, whether the induced MNGCs will lead to material degradation or encapsulation over the long term requires further investigation. CLINICAL RELEVANCE: The data presented are of great clinical interest, as they contribute to the ongoing discussion concerning to what extent an implanted material should be integrated versus serving only as a barrier membrane.

Construction of a cDNA library for miniature pig mandibular deciduous molars.

BACKGROUND: The miniature pig provides an excellent experimental model for tooth morphogenesis because its diphyodont and heterodont dentition resembles that of humans. However, little information is available on the process of tooth development or the exact molecular mechanisms controlling tooth development in miniature pigs or humans. Thus, the analysis of gene expression related to each stage of tooth development is very important. RESULTS: In our study, after serial sections were made, the development of the crown of the miniature pigs' mandibular deciduous molar could be divided into five main phases: dental lamina stage (E33-E35), bud stage (E35-E40), cap stage (E40-E50), early bell stage (E50-E60), and late bell stage (E60-E65). Total RNA was isolated from the tooth germ of miniature pig embryos at E35, E45, E50, and E60, and a cDNA library was constructed. Then, we identified cDNA sequences on a large scale screen for cDNA profiles in the developing mandibular deciduous molars (E35, E45, E50, and E60) of miniature pigs using Illumina Solexa deep sequencing. Microarray assay was used to detect the expression of genes. Lastly, through Unigene sequence analysis and cDNA expression pattern analysis at E45 and E60, we found that 12 up-regulated and 15 down-regulated genes during the four periods are highly conserved genes homologous with known Homo sapiens genes. Furthermore, there were 6 down-regulated and 2 up-regulated genes in the miniature pig that were highly homologous to Homo sapiens genes compared with those in the mouse. CONCLUSION: Our results not only identify the specific transcriptome and cDNA profile in developing mandibular deciduous molars of the miniature pig, but also provide useful information for investigating the molecular mechanism of tooth development in the miniature pig.

Spatial and temporal expression of c-Kit in the development of the murine submandibular gland.

The c-Kit pathway is important in the development of many mammalian cells and organs and is indispensable for the development of hematopoiesis, melanocytes, and primordial germ cells. Loss-of-function mutations in c-Kit lead to perinatal death in mouse embryos. Previously, c-Kit has been used as one of salivary epithelial stem or progenitor cell markers in mouse, its specific temporo-spatial expression pattern and function in developing murine submandibular gland (SMG) is still unclear. Here we used quantitative real-time PCR, in situ hybridization, and immunohistochemistry analysis to detect c-Kit expression during the development of the murine SMG. We found that c-Kit was expressed in the epithelia of developing SMGs from embryonic day 11.5 (E11.5; initial bud stage) to postnatal day 90 (P90; when the SMG is completely mature). c-Kit expression in the end bud epithelium increased during prenatal development and then gradually decreased after birth until its expression was undetectable in mature acini at P30. Moreover, c-Kit was expressed in the SMG primordial cord at the initial bud, pseudoglandular, canacular, and terminal end bud stages. c-Kit was also expressed in the presumptive ductal cells adjacent to the developing acini. By the late terminal end bud stage on P14, c-Kit expression could not be detected in ductal cells. However, c-Kit expression was detected in ductal cells at P30, and its expression had increased dramatically at P90. Taken together, these findings describe the spatial and temporal expression pattern of c-Kit in the developing murine SMG and suggest that c-Kit may play roles in epithelial histo-morphogenesis and in ductal progenitor cell homeostasis in the SMG.

Histological and ultrastructural characterization of developing miniature pig salivary glands.

Salivary glands are a classic model of organ development and differentiation. Miniature pigs are considered as a unique animal model for salivary gland researchers in the fields of gene transfer, radiation damage, and functional reconstruction. However, there is little information about the development of miniature pig salivary glands. The present article was designed to study the developmental stages of salivary glands in miniature pigs using histological and ultrastructural methods. Sections from E40, E60, E80, E95 embryos, and P0 pups were stained with hematoxylin-eosin, Alcian blue, or periodic acid-schiff. Selected specimens were also processed for electron microscopy. The development of the miniature pig salivary glands can be divided into five different stages that refer to the stages of the developing mouse submandibular gland. The histological characteristics of the miniature pig salivary glands at different developmental stages were synchronously verified at the ultrastructural level. Interestingly, the development of the miniature pig parotid gland trailed that of the submandibular gland by approximately 15 days. Our study provides first-hand data regarding the morphological organogenesis of salivary glands in the miniature pig and provides a foundation for further research on this model.

Disruption of Smad4 in odontoblasts causes multiple keratocystic odontogenic tumors and tooth malformation in mice.

Keratocystic odontogenic tumors (KCOTs) are cystic epithelial neoplasias with a high recurrence rate. However, the molecular mechanisms underlying the initiation and progression of KCOTs are still largely unknown. Here, we show that specific ablation of Smad4 in odontoblasts unexpectedly resulted in spontaneous KCOTs in mice. The mutant mice exhibited malformed teeth characterized by fractured incisors and truncated molar roots. These abnormalities were mainly caused by disrupted odontoblast differentiation that led to irregular dentin formation. The cystic tumors arising from the reactivation of epithelial rests of Malassez (ERM), in which Smad4 remained intact, proliferated and formed stratified and differentiated squamous epithelia that exhibited a dramatic upregulation of Hedgehog signaling. Odontoblasts, which are responsive to transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) signals, may produce signal molecules to inhibit the activation of ERM. Indeed, we observed a downregulation of BMP signals from Smad4 mutant odontoblasts to the adjacent Hertwig's epithelial root sheath (HERS). Intriguingly, KCOTs frequently emerged from Smad4-deficient ERM in keratinocyte-specific Smad4 knockout mice, suggesting a novel mechanism in which reciprocal TGF-beta/BMP signaling between odontoblasts and HERS was required for tooth root development and suppression of KCOT formation. These findings provide insight into the genetic basis underlying KCOTs and have important implications for new directions in KCOT treatment.

[Tooth regeneration--dream to reality].

Tooth or dentition missing compromises human health physically and psychiatrically. Although several prosthesis methods are used to restore tooth loss, these restorations are still non-biological methods. It is a dream for human being to regenerate a real tooth for hundreds years. There are two ways to regenerate the tooth. One is application of conventional tissue engineering techniques including seed cells and scaffold. The other is regeneration tooth using dental epithelium and dental mesenchymal cells based on the knowledge of tooth initiation and development. Marked progress has been achieved in these two ways, while there is still a long way to go. Recently a new concept has been proposed for regeneration of a biological tooth root based on tooth-related stem cells and tissue engineering technique. A biological tooth root has been regenerated in swine. It may be a valuable method for restoration of tooth loss before successful whole tooth regeneration. A latest research showed that a subpopulation in bone marrow cells can give rise to ameloblast-like cells when mixed with embryonic epithelium and reassociation with integrated mesenchyme, which may provide a new seed cell source for tooth regeneration.