Dental pulp stem cells and banking of teeth as a lifesaving therapeutic vista

Exfoliated deciduous or an extracted healthy adult tooth can be used to harvest, process, and cryogenically preserve dental pulp stem cells. Future stem cell-based regenerative medicine methods could benefit significantly from these mesenchymal stem cells. Teeth serve as a substantial source of mesenchymal stem cells, otherwise disposed of as medical waste. Care should be taken to store this treasure trove of stem cells. Collective responsibility of patients, dentists, and physicians is necessary to ensure that this valuable resource is not wasted and that every possible dental pulp stem cell is available for use in the future. The dental pulp stem cells (DPSC) inside teeth represent a significant future source of stem cells for regenerative medicine procedures. This review describes the ontogeny, the laboratory processing and collection, and isolation methods of DPSC. This review also discusses currently available stem cell banking facilities and their potential use in regenerative medicine procedures in dental and general medical applications in the future.     

Skeletal stem cells and bone regeneration: translational strategies from bench to clinic

Clinical imperatives for new bone to replace or restore the function of traumatized or bone lost as a consequence of age or disease has led to the need for therapies or procedures to generate bone for skeletal applications. Tissue regeneration promises to deliver specifiable replacement tissues and the prospect of efficacious alternative therapies for orthopaedic applications such as non-union fractures, healing of critical sized segmental defects and regeneration of articular cartilage in degenerative joint diseases. In this paper we review the current understanding of the continuum of cell development from skeletal stem cells, osteoprogenitors through to mature osteoblasts and the role of the matrix microenvironment, vasculature and factors that control their fate and plasticity in skeletal regeneration. Critically, this review addresses in vitro and in vivo models to investigate laboratory and clinical based strategies for the development of new technologies for skeletal repair and the key translational points to clinical success. The application of developmental paradigms of musculoskeletal tissue formation specifically, understanding developmental biology of bone formation particularly in the adult context of injury and disease will, we propose, offer new insights into skeletal cell biology and tissue regeneration allowing for the critical integration of stem cell science, tissue engineering and clinical applications. Such interdisciplinary, iterative approaches will be critical in taking patient aspirations to clinical reality.     

Periodontal regeneration: a challenge for the tissue engineer?

Periodontitis affects around 15 per cent of human adult populations. While periodontal treatment aimed at removing the bacterial cause of the disease is generally very successful, the ability predictably to regenerate the damaged tissues remains a major unmet objective for new treatment strategies. Existing treatments include the use of space-maintaining barrier membranes (guided tissue regeneration), use of graft materials, and application of bioactive molecules to induce regeneration, but their overall effects are relatively modest and restricted in application. The periodontal ligament is rich in mesenchymal stem cells, and the understanding of the signalling molecules that may regulate their differentation has increased enormously in recent years. Applying these principles for the development of new tissue engineering strategies for periodontal regeneration will require further work to determine the efficacy of current experimental preclinical treatments, including pharmacological application of growth factors such as bone morphogenetic proteins (BMPs) or Wnts, use of autologous stem cell reimplantation strategies, and development of improved biomaterial scaffolds. This article describes the background to this problem, addresses the current status of periodontal regeneration, including the background biology, and discusses the potential for some of these experimental therapies to achieve the goal of clinically predictable periodontal regeneration.     

Mesenchymal stem cells,the secretome and biomaterials: Regenerative medicine application

Mesenchymal stem cells (MSCs) are multipotent cells usually isolated from bone marrow, endometrium, adipose tissues, skin, and dental pulp. MSCs played a crucial role in regenerative therapy and have been introduced as an interdisciplinary field between cell biology and material science. Recently, MSCs have been widely explored for their application in regenerative medicine and COVID-19 treatment. Different approaches to evaluate the future of biomaterials and stem cell properties have been developed. However, misconceptions and ethical issues still exist, such as MSCs being non-angiogenic, anti-apoptotic, and immunoregulatory competencies. Embryonic stem cells isolation primarily requires the consent of donors and can include the killing of fertilized eggs. These issues generate questions related to ethical and moral issues. However, MSCs have gained considerable attention for tissue regeneration owing to their differentiation ability with immunomodulatory effects. They are capable of secreting a broad range of biomolecules such as proteins, nucleic acids, exosomes, microRNAs, and membrane vesicles, collectively known as secretomes. Secretomes are released in response to the surrounding microenvironment. In this article, we briefly address topics related to the therapeutic potential of MSCs as an advanced approach in the field of regenerative medicine and various perspectives.     

Distribution of nestin protein: immunohistochemical study in enteric plexus of rat duodenum

The intermediate nestin filament is expressed in neural stem cells, neuroectodermal tumors and various adult tissues under situations that reproduce developmental phases, e.g., physiological renewal of certain cell types, tissue regeneration, and healing or revascularization. In the human gastrointestinal tract, nestin has been reported in glial cells and interstitial cells of Cajal. We examined by immunohistochemistry the appearance and distribution of nestin protein in enteric ganglia of rat duodenum. Through the myenteric and submucosal plexuses, a high number of nestin-positive cells were visualized in this specie. The nestin-positive cells were smaller and more numerous than enteric neurons. They were present both within and around ganglia. The results of this study suggest that the rat enteric glial cells (EGCs) are rich in nestin, a protein usually associated with dividing or migrating cells and the dynamic reorganization of nestin filaments during the cell cycle. EGCs could function as enteric stem cells.     

Dental neuroplasticity,neuro-pulpal interactions,and nerve regeneration

This review covers current information about the ability of dental nerves to regenerate and the role of tooth pulp in recruitment of regenerating nerve fibers. In addition, the participation of dental nerves in pulpal injury responses and healing is discussed, especially concerning pulp regeneration and reinnervation after tooth replantation. The complex innervation of teeth is highly asymmetric and guided towards specific microenvironments along blood vessels or in the crown pulp and dentin. Pulpal products such as nerve growth factor are distributed in the same asymmetric gradients as the dentinal sensory innervation, suggesting regulation and recruitment of those nerve fibers by those specific factors. The nerve fibers have important effects on pulpal blood flow and inflammation, while their sprouting and cytochemical changes after tooth injury are in response to altered pulpal cytochemistry. Thus, their pattern and neuropeptide intensity are indicators of pulp status, while their local actions continually affect that status. When denervated teeth are injured, either by pulp exposure on the occlusal surface or by replantation, they have more pulpal necrosis than occurs for innervated teeth. However, small pulp exposures on the side of denervated crowns or larger lesions in germ-free animals can heal well, showing the value of postoperative protection from occlusal trauma or from infection. Current ideas about dental neuroplasticity, neuro-pulpal interactions, and nerve regeneration are related to the overall topics of tooth biomimetics and pulp/dentin regeneration.     

Production of mesenchymal stem cell derived-secretome as cell-free regenerative therapy and immunomodulation: A biomanufacturing perspective

The potential of mesenchymal stem cells (MSCs) in regenerative medicine has been largely known due to their capability to induce tissue regeneration in vivo with minimum inflammation during implantation. This adult stem cell type exhibit unique features of tissue repair mechanism and immune modulation mediated by their secreted factors, called secretome. Recently, the utilization of secretome as a therapeutic agent provided new insight into cell-free therapy. Nevertheless, a sufficient amount of secretome is necessary to realize their applications for translational medicine which required a proper biomanufacturing process. Several factors related to their production need to be considered to produce a clinical-grade secretome as a biological therapeutic agent. This viewpoint highlights the current challenges and considerations during the biomanufacturing process of MSCs secretome.     

Processing of soft pupae and uneclosed pharate adults of Drosophila for scanning electron microscopy

For over four decades, scanning electron microscopy (SEM) has been used in research involving Drosophila genetics and developmental biology. It allows for observation and documentation of the gross morphology of exoskeletal structures as well as their characterization at very high resolution. In most cases, SEM in Drosophila has been limited to imaging adult heads, thoraces, appendages, and embryos, as these structures are relatively hard and/or easy to process for SEM. In contrast, the structures of the pharate adult stages are difficult to prepare for SEM because their integument is quite soft, they are extremely dirty and they are resistant to the usual processing methods. Here, we present an innovative method to prepare these types of structures. Our protocol efficiently removes extraneous material originating from the exuvial fluid of pharate adults and uses a hydrophobic expansion step to keep the soft exoskeleton of the body inflated. In addition to using immersion fixation, it utilizes fixation within the body that occurs via a reaction between osmium tetroxide and alcohols that are infiltrated into the body during a hydrophobic expansion step. This novel approach results in a properly inflated integument that retains its shape in subsequent procedures. Our method provides a useful, general alternative for processing difficult samples, including soft, biological "whole-mount" specimens and samples that are extremely dirty or resistant to fixative penetration.     

Degeneration and regeneration of ganglion cell axons

The retino-tectal system has been used to study developmental aspects of axon growth, synapse formation and the establishment of a precise topographic order as well as degeneration and regeneration of adult retinal ganglion cell (RGC) axons after axonal lesion. This paper reviews some novel findings that provide new insights into the mechanisms of developmental RGC axon growth, pathfinding, and target formation. It also focuses on the cellular and molecular cascades that underlie RGC degeneration following an axonal lesion and on some therapeutic strategies to enhance survival of axotomized RGCs in vivo. In addition, this review deals with problems related to the induction of regeneration after axonal lesion in the adult CNS using the retino-tectal system as model. Different therapeutic approaches to promote RGC regeneration and requirements for specific target formation of regenerating RGCs in vitro and in vivo are discussed.     

Isolation of stem cells from adult rat kidneys

The kidney has an inherent ability for recovery and regeneration following acute damage. However, there has been much contention as to the source of regenerating renal cells. The aim of this study was to isolate and characterize these cells. Normal rat kidneys were minced and cells were isolated with collagenase I and were cultured in an expansion medium. Adherent cells were isolated and expanded for more than 120 days in vitro. These cells had the potential of trans-lineage differentiation into neural cells, adipocytes and osteocytes. These cells also expressed Nucleostemin, Cyclin D1, Notch1 and Survivin which are commonly expressed in stem cells. The results of the current work show that the adult kidney contains a population of multipotent stem cells.     

Single-cell sequencing analysis reveals the molecular mechanism of promotion of SCAP proliferation upon AZD2858 treatment

The Wnt/β-catenin signaling pathway is the main target of tooth regeneration regulation. Treatment of cells with AZD2858 stimulates the Wnt/β-catenin signaling pathway, yet the function of this pathway in tooth regeneration remains unclear. Here, we found that AZD2858 promotes the accumulation of β-catenin in the nuclei of stem cells from the apical papilla (SCAPs) and enhances cell proliferation. Single-cell sequencing was performed on SCAPs treated with AZD2858. Eight clusters were identified, namely SCAPs-CNTNAP2, SCAPs-DTL, SCAPs-MYH11, SCAPs-MKI67, SCAPs-CXCL8, SCAPs-TPM2, SCAPs-IFIT2 and SCAPs-NEK10. The pseudo-time trajectory analysis showed that AZD2858 enhanced the evolution of SCAPs from SCAPs-TMP2 clusters to SCAPs-MYH11, SCAPs-CNTNAPs and SCAPs-NEK10 clusters via up-regulation of PRKCA, SMURF2, MAGI2, RBMS3, EXT1, CAMK2D, PLCB4, and PLCB1. These results demonstrate that AZD2858 enhances the proliferation of SCAPs-TPM2 cluster by activating the non-canonical Wnt/β-catenin signaling pathway.     

MSCs-exosomes in regeneration medicine: Current evidence and future perspectives

Exosomes, especially from mesenchymal stem cells, have attracted extensive attention in regeneration medicine. Mesenchymal stem cells derived exosomes (MSCs-exosomes) have shown anti-inflammatory, anti-oxidant, anti-apoptosis and tissue regeneration effects in a variety of tissue injury repair models. MSCs-exosomes hold many excellent properties such as low immunogenicity, biocompatibility, and targeting capability. With the in-depth study on the generation and function of exosomes, MSCs-exosomes are considered to be the bright stars in the field of regenerative medicine. However, there are still many obstacles to overcome in terms of exosomes isolation, clinical trials and safety evaluation. In this article, what we should focus on about MSCs-exosomes in regeneration medicine will be discussed.     

Quantitative histological and ultrastructural features of opercula of normally erupting human teeth

Tooth eruption across the mucosa in humans has been studied rarely, although there are disturbances of eruption that are attributed specifically to failure of the supraosseous eruptive migration. The aim of this study was to analyze the soft tissues covering normally erupting teeth so as to get an insight into the supraosseous phase of tooth eruption and to provide the basis for comparison with cases of eruption disturbances. Six opercula covering normally erupting permanent molars (primary opercula) and six of succedaneous teeth (secondary opercula) were surgically removed from 10 patients aged 7.5-17.5 years. Specimens were examined light and electron microscopically and analyzed morphometrically. All opercula contained strands and islands of odontogenic epithelium, prominent numbers of high endothelial venules, nerves, and mast cells. Nerves comprised normally structured, 1.5-3.5 microm thick myelinated (Adelta) and thinner unmyelinated (C) fibers. In primary opercula, the proportions of blood vessels and nerves were three- and sevenfold higher than the respective values for the secondary opercula. Furthermore, primary opercula contained multinucleated, fibroblast-like giant cells that were not observed in secondary opercula. As all teeth under investigation were erupting normally, neither the presence of the giant cells nor the atypical proportions of blood vessels and nerves appeared to be decisive in the eruption process. These conspicuous tissue components of opercula seem merely to accompany the eruptive tooth movement.     

螺旋锥齿轮拟合齿面啮合特性分析方法

已知齿面测量的采样点，利用微分几何及Ferguson参数样条曲面原理，建立了螺旋锥齿轮齿面拟合的数学模型。根据优化求极值复合形方法和共轭理论，通过变定义域，快速求解齿轮副拟合齿面接触点，并运用V—H接触区调整方法，研究了拟合齿面接触迹、接触区和运动误差曲线的求解算法，绘制出相关图形并给出了实例。     

Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction

An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF-β1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF-β1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF-β1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF.     

基于特征映射的义齿表面三维变形设计方法

义齿表面设计是牙科计算机辅助设计和制造系统的一个重要环节,设计的义齿形状必须符合患者的口腔咬合环境并能保持牙齿的拓扑结构特征。针对这一问题,在此提出一种基于特征映射的义齿表面三维变形设计方法。该方法主要思想是首先识别预备体与同名标准牙的重要特征点,采用径向基函数建立两者的特征映射关系,再通过对应特征点的位置对齐和表面的插值变形,实现义齿表面的变形设计。下颌第一磨牙嵌体的设计案例结果表明,该方法设计程序简单,标准牙曲面变形合理,能有效地避免曲面失真,最终生成的嵌体与预备体匹配良好,为牙科计算机辅助设计和制造系统的临床应用提供一种新的方法。     

Morphological characterization of the progenitor blood cells in canine and feline umbilical cord

The umbilical cord blood (UCB) is an important source of hematopoietic stem cells with great deal of interest in regenerative medicine. The UCB cells have been extensively studied as an alternative to the bone marrow transplants. The challenge is to define specific methods to purify and characterize these cells in different animal species. This study is aimed at morphological characterization of progenitor cells derived from UCB highlighting relevant differences with peripheral blood of adult in dog and cats. Therefore, blood was collected from 18 dogs and 5 cats' umbilical cords from fetus in various developmental stages. The mononuclear cells were separated using the gradient of density Histopaque-1077. Characterization of CD34+ cells was performed by flow cytometric analysis and transmission electron microscopy. Granulocytes (ancestry of the basophiles, eosinophiles, and neutrophiles) and agranulocytes (represented by immature lymphocytes) were identified. We showed for the first time the ultrastructural features of cat UCB cells.     

Microscopic overview of crinoid regeneration.

Crinoids are well known for their striking regenerative potential and can rapidly and completely regenerate arms lost following self-induced or traumatic amputation. Thus they provide a valuable experimental model for investigation of the regenerative process from the macroscopic to the molecular level. In these last years we have studied in detail the overall process of arm regeneration in the comatulid Antedon mediterranea. This phenomenon can be described on the whole as a typical blastemal regeneration in which new structures develop from migratory pluripotential, actively proliferating cells in the presence of presumptive regulatory factors. The overall process can be subdivided into three main phases: a repair phase, an early regenerative phase, and an advanced regenerative phase, whose crucial aspects are related to common fundamental mechanisms such as cell migration and proliferation, intervention of stem cells and/or dedifferentiated cells, contribution of putative growth factors, particularly in terms of specific neurally derived factors, and mechanisms of pattern formation. This article focuses on the main aspects of the phenomenon and gives a brief account of the most recent and relevant results. Our approach employs classical methods of light (LM) and electron (TEM and SEM) microscopy, immunocytochemistry, and histofluorescence on experimentally induced arm regenerations of standard or abnormal type obtained in significantly different experimental conditions, including extreme mutilations (explants) or exposure to pseudo-estrogenic environmental contamination.