Engineered neural tissue with Schwann cell differentiated human dental pulp stem cells: potential for peripheral nerve repair?

Despite the spontaneous regenerative capacity of the peripheral nervous system, large gap peripheral nerve injuries (PNIs) require bridging strategies. The limitations and suboptimal results obtained with autografts or hollow nerve conduits in the clinic urge the need for alternative treatments. Recently, we have described promising neuroregenerative capacities of Schwann cells derived from differentiated human dental pulp stem cells (d-hDPSCs) in vitro. Here, we extended the in vitro assays to show the pro-angiogenic effects of d-hDPSCs, such as enhanced endothelial cell proliferation, migration and differentiation. In addition, for the first time we evaluated the performance of d-hDPSCs in an in vivo rat model of PNI. Eight weeks after transplantation of NeuraWrap™ conduits filled with engineered neural tissue (EngNT) containing aligned d-hDPSCs in 15-mm rat sciatic nerve defects, immunohistochemistry and ultrastructural analysis revealed ingrowing neurites, myelinated nerve fibres and blood vessels along the construct. Although further research is required to optimize the delivery of this EngNT, our findings suggest that d-hDPSCs are able to exert a positive effect in the regeneration of nerve tissue in vivo. Copyright © 2017 John Wiley & Sons, Ltd.

Label-free mapping of microstructural organisation in self-aligning cellular collagen hydrogels using image correlation spectroscopy.

Hydrogels have emerged as promising biomaterials for regenerative medicine. Despite major advances, tissue engineers have faced challenges in studying the complex dynamics of cell-mediated hydrogel remodelling. Second harmonic generation (SHG) microscopy has been a pivotal tool for non-invasive visualization of collagen type I hydrogels. By taking into account the typical polarization SHG effect, we recently proposed an alternative image correlation spectroscopy (ICS) model to quantify characteristics of randomly oriented collagen fibrils. However, fibril alignment is an important feature in many tissues that needs to be monitored for effective assembly of anisotropic tissue constructs. Here we extended our previous approach to include the orientation distribution of fibrils in cellular hydrogels and show the power of this model in two biologically relevant applications. Using a collagen hydrogel contraction assay, we were able to capture cell-induced hydrogel modifications at the microscopic scale and link these to changes in overall gel dimensions over time. After 24h, the collagen density was about 3 times higher than the initial density, which was of the same order as the decrease in hydrogel area. We also showed that the orientation parameters recovered from our automated ICS model match values obtained from manual measurements. Furthermore, regions axial to cellular processes aligned at least 1.5 times faster compared with adjacent zones. Being able to capture minor temporal and spatial changes in hydrogel density and collagen fibril orientation, we demonstrated the sensitivity of this extended ICS model to deconstruct a complex environment and support its potential for tissue engineering research. STATEMENT OF SIGNIFICANCE: It is generally accepted that looking beyond bulk hydrogel composition is key in understanding the mechanisms that influence the mechanical and biological properties of artificial tissues. In this manuscript, we performed label-free non-invasive imaging and extended a robust automated analysis method to characterize the microstructural organisation of cellular hydrogel systems. We underpin the sensitivity of this technique by capturing minor changes in collagen density and fibril orientation in biologically relevant systems over time. Therefore, we believe that this method is applicable in fundamental cell-matrix research and has high-throughput potential in screening arrays of hydrogel scaffolds, making it an interesting tool for future tissue engineering research.

Sensory innervation around immediately vs. delayed loaded implants: a pilot study.

Although neurophysiological and psychophysical proof of osseoperception is accumulating, histomorphometric evidence for the neural mechanisms of functional compensation following immediate and delayed implant loading is still lacking. For this randomized split-mouth study, six mongrel dogs randomly received one of four treatment protocols at 36 implant-recipient sites over 16 weeks (third maxillary incisor, third and fourth mandibular premolar): immediate implant placement and immediate loading (IIP+IL); delayed implant placement and delayed loading (DIP+DL); delayed implant placement and immediate loading (DIP+IL); and natural extraction socket healing (control). Histomorphometry was performed in the peri-implant bone and soft tissues within 300 µm around the implants. Immunocytochemistry and transmission electron microscopy were used to confirm the presence of neural structures and to reveal their ultrastructural characteristics, respectively. Myelinated nerve fibres densely populated the peri-implant crestal gingival and apical regions, although they were also identified in the woven bone and in the osteons near the implant threads. Compared with the control group in the mandible, the group that received IIP+IL showed a higher innervation (in N⋅mm⁻², 5.94 ± 1.12 vs. 3.15 ± 0.63, P<0.001) and smaller fibre diameter (in µm, 1.37 ± 0.05 vs. 1.64 ± 0.13, P=0.016), smaller axon diameter (in µm, 0.89 ± 0.05 vs. 1.24 ± 0.10, P=0.009) and g-ratio (0.64 ± 0.04 vs. 0.76 ± 0.05, P<0.001) in the middle region around the implants. Compared with DIP+IL in the mandible, IIP+IL had a higher nerve density (in N⋅mm⁻², 13.23 ± 2.54 vs. 9.64 ± 1.86, P=0.027), greater fibre diameter (in µm, 1.32 ± 0.02 vs. 1.20 ± 0.04, P=0.021), greater axon diameter (in µm, 0.92 ± 0.01 vs. 0.89 ± 0.03, P=0.035) and lower g-ratio (0.69 ± 0.01 vs. 0.74 ± 0.01, P=0.033) in the apical region around the implants. It may be assumed that the treatment protocol with IIP+IL is the preferred method to allow optimized peri-implant re-innervation, but further functional measurements are still required.

Neurogenic maturation of human dental pulp stem cells following neurosphere generation induces morphological and electrophysiological characteristics of functional neurons.

Cell-based therapies are emerging as an alternative treatment option to promote functional recovery in patients suffering from neurological disorders, which are the major cause of death and permanent disability. The present study aimed to differentiate human dental pulp stem cells (hDPSCs) toward functionally active neuronal cells in vitro. hDPSCs were subjected to a two-step protocol. First, neuronal induction was acquired through the formation of neurospheres, followed by neuronal maturation, based on cAMP and neurotrophin-3 (NT-3) signaling. At the ultrastructural level, it was shown that the intra-spheral microenvironment promoted intercellular communication. hDPSCs grew out of the neurospheres in vitro and established a neurogenic differentiated hDPSC culture (d-hDPSCs) upon cAMP and NT-3 signaling. d-hDPSCs were characterized by the increased expression of neuronal markers such as neuronal nuclei, microtubule-associated protein 2, neural cell adhesion molecule, growth-associated protein 43, synapsin I, and synaptophysin compared with nondifferentiated hDPSCs. Enzyme-linked immunosorbent assay demonstrated that the secretion of brain-derived neurotrophic factor, vascular endothelial growth factor, and nerve growth factor differed between d-hDPSCs and hDPSCs. d-hDPSCs acquired neuronal features, including multiple intercommunicating cytoplasmic extensions and increased vesicular transport, as shown by the electron microscopic observation. Patch clamp analysis demonstrated the functional activity of d-hDPSCs by the presence of tetrodotoxin- and tetraethyl ammonium-sensitive voltage-gated sodium and potassium channels, respectively. A subset of d-hDPSCs was able to fire a single action potential. The results reported in this study demonstrate that hDPSCs are capable of neuronal commitment following neurosphere formation, characterized by distinct morphological and electrophysiological properties of functional neuronal cells.

Peri-implant bone innervation: histological findings in humans.

PURPOSE: The aim of the present study was to describe nerve fibres around osseointegrated implants in humans. MATERIALS AND METHODS: Twelve mechanically failed implants, retrieved from 10 patients were collected from three dental centres over a period of 5 years. After implant removal, decalcified semi-thin sections (0.5 µm) were stained with thionic methylene blue for light microscopic analysis. In addition, an ultrastructural analysis was performed on serial ultra-thin sections (0.06 µm) using transmission electron microscopy. RESULTS: Both myelinated and unmyelinated nerve fibres could be identified inside the Haversian canals of the osteonal bone near the implant threads. Myelinated fibres were also located at the woven bone around the implant. However, no differentiated nerve endings could be observed around the implants. CONCLUSIONS: This study shows the presence of nerve fibres in human peri-implant bone. Previous studies in animals showed that those fibres participate in the process of bone modelling and remodelling. Yet, the role of peri-implant bone innervation in the osseoperception phenomenon cannot be ruled out since the mechanism of mechanoreception in bone is not fully understood.

Presurgical CBCT assessment of maxillary neurovascularization in relation to maxillary sinus augmentation procedures and posterior implant placement.

PURPOSE: To provide more information to clinicians planning sinus grafting and maxillofacial surgical interventions, the present study evaluated the prevalence, diameter and location of the superior alveolar canals (SAC) using CBCT images. METHODS: The maxillary sinus CBCT scans (i-CAT Classic(®), ISI, USA) of 100 adult patients (67 women and 33 men) aged 20-79 years [mean (SD) 40 (15)] were examined. A dentomaxillofacial radiologist observed the SAC based on CBCT image data and more specifically the parasagittal views to assess SAC's diameter and location. RESULTS: The anterior and posterior SAC, double ASAC, intraosseous anastomoses and the extension of the anterior SAC to the piriform aperture were observed in 100, 73, 24.5, 38.5 and 84 % of the cases, respectively. The anastomosis was located between canine and first premolar in 43 % of the cases. The SAC diameters were in 80 % of the cases ≤1 mm, remaining canals had a diameter between 1 and 2 mm. The distance of the SAC to the alveolar crest ranged between 2.42 and 44.6 mm. The anterior SAC was more prevalent in the upper (53 %) and middle (44 %) thirds of the maxillary sinus, while the posterior SAC was more prevalent in the middle (36 %) and lower thirds (64 %). The distance was significantly bigger in men in some tooth positions. CONCLUSIONS: Based on the present findings, one-fifth of the patients may have a diameter of the SAC >1 mm, large enough to cause bleeding and/or paraesthesia. CBCT imaging may assist surgeons to plan grafting and osteotomy procedures, while avoiding these neurovascular structures.

Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis.

Mesenchymal stem cells or multipotent stromal cells (MSCs) have initially captured attention in the scientific world because of their differentiation potential into osteoblasts, chondroblasts and adipocytes and possible transdifferentiation into neurons, glial cells and endothelial cells. This broad plasticity was originally hypothesized as the key mechanism of their demonstrated efficacy in numerous animal models of disease as well as in clinical settings. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly caused by the multitude of bioactive molecules secreted by these remarkable cells. Numerous angiogenic factors, growth factors and cytokines have been discovered in the MSC secretome, all have been demonstrated to alter endothelial cell behavior in vitro and induce angiogenesis in vivo. As a consequence, MSCs have been widely explored as a promising treatment strategy in disorders caused by insufficient angiogenesis such as chronic wounds, stroke and myocardial infarction. In this review, we will summarize into detail the angiogenic factors found in the MSC secretome and their therapeutic mode of action in pathologies caused by limited blood vessel formation. Also the application of MSC as a vehicle to deliver drugs and/or genes in (anti-)angiogenesis will be discussed. Furthermore, the literature describing MSC transdifferentiation into endothelial cells will be evaluated critically.

Human dental pulp stem cells can differentiate into Schwann cells and promote and guide neurite outgrowth in an aligned tissue-engineered collagen construct in vitro.

In the present study, we evaluated the differentiation potential of human dental pulp stem cells (hDPSCs) toward Schwann cells, together with their functional capacity with regard to myelination and support of neurite outgrowth in vitro. Successful Schwann cell differentiation was confirmed at the morphological and ultrastructural level by transmission electron microscopy. Furthermore, compared to undifferentiated hDPSCs, immunocytochemistry and ELISA tests revealed increased glial marker expression and neurotrophic factor secretion of differentiated hDPSCs (d-hDPSCs), which promoted survival and neurite outgrowth in 2-dimensional dorsal root ganglia cultures. In addition, neurites were myelinated by d-hDPSCs in a 3-dimensional collagen type I hydrogel neural tissue construct. This engineered construct contained aligned columns of d-hDPSCs that supported and guided neurite outgrowth. Taken together, these findings provide the first evidence that hDPSCs are able to undergo Schwann cell differentiation and support neural outgrowth in vitro, proposing them to be good candidates for cell-based therapies as treatment for peripheral nerve injury.

Angiogenic properties of human dental pulp stem cells.

Angiogenesis, the formation of capillaries from pre-existing blood vessels, is a key process in tissue engineering. If blood supply cannot be established rapidly, there is insufficient oxygen and nutrient transport and necrosis of the implanted tissue will occur. Recent studies indicate that the human dental pulp contains precursor cells, named dental pulp stem cells (hDPSC) that show self-renewal and multilineage differentiation capacity. Since these cells can be easily isolated, cultured and cryopreserved, they represent an attractive stem cell source for tissue engineering. Until now, only little is known about the angiogenic abilities and mechanisms of the hDPSC. In this study, the angiogenic profile of both cell lysates and conditioned medium of hDPSC was determined by means of an antibody array. Numerous pro-and anti-angiogenic factors such as vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1) and endostatin were found both at the mRNA and protein level. hDPSC had no influence on the proliferation of the human microvascular endothelial cells (HMEC-1), but were able to significantly induce HMEC-1 migration in vitro. Addition of the PI3K-inhibitor LY294002 and the MEK-inhibitor U0126 to the HMEC-1 inhibited this effect, suggesting that both Akt and ERK pathways are involved in hDPSC-mediated HMEC-1 migration. Antibodies against VEGF also abolished the chemotactic actions of hDPSC. Furthermore, in the chicken chorioallantoic membrane (CAM) assay, hDPSC were able to significantly induce blood vessel formation. In conclusion, hDPSC have the ability to induce angiogenesis, meaning that this stem cell population has a great clinical potential, not only for tissue engineering but also for the treatment of chronic wounds, stroke and myocardial infarctions.

Histomorphological study of myelinated nerve fibres in the periodontal ligament of human canine.

OBJECTIVE: This study aims to describe the human periodontal ligament (PDL) using serial sections, with a focus on mechanoreceptor distribution and morphology. MATERIALS AND METHODS: One permanent lower canine with surrounding PDL and alveolar bone tissues was retrieved from a human cadaver. After being embedded into paraffin block, the canine was horizontally cut in 6 µm thick serial sections. At root levels of 0.3, 1.5, 3, 4.5 and 6 mm from the apex, five slices each level were evaluated. Immunocytochemisty was performed on the same serial sections, enabling a more reliable description of neural structures. RESULTS: The distribution of myelinated fibres varied from apical to coronal level, with a total number of 38 at 0.3 mm from the apex, 25 at 1.5 mm, 25 at 3 mm, 31 at 4.5 mm and 32 at 6 mm. At all times, mesial and buccal regions were typically more densely innervated (p < 0.01) except at the 3 mm level. The average density of myelinated nerve fibres increased by arriving closer to the apex. However, the average diameter did not show any significant differences amongst quadrants or root levels (p > 0.05). The average diameter of myelinated fibres varied between 5.3-7.8 µm. Grouped myelinated axons were twice as common as isolated ones, with the innervation being rather close to the alveolar bone. Isolated myelinated axons showed a tendency to group around large blood vessels. CONCLUSION: The present results add to the understanding of human PDL innervation, indicating dense innervations by myelinated nerve fibres in close proximity to collagen fibres and alveolar bone. It also reveals that apical as well as mesial and buccal sites of the human canine are more densely innervated.

A comparative evaluation of Cone Beam Computed Tomography (CBCT) and Multi-Slice CT (MSCT) Part I. On subjective image quality.

AIMS: To compare image quality and visibility of anatomical structures in the mandible between five Cone Beam Computed Tomography (CBCT) scanners and one Multi-Slice CT (MSCT) system. MATERIALS AND METHODS: One dry mandible was scanned with five CBCT scanners (Accuitomo 3D, i-CAT, NewTom 3G, Galileos, Scanora 3D) and one MSCT system (Somatom Sensation 16) using 13 different scan protocols. Visibility of 11 anatomical structures and overall image noise were compared between CBCT and MSCT. Five independent observers reviewed the CBCT and the MSCT images in the three orthographic planes (axial, sagittal and coronal) and assessed image quality on a five-point scale. RESULTS: Significant differences were found in the visibility of the different anatomical structures and image noise level between MSCT and CBCT and among the five CBCT systems (p=0.0001). Delicate structures such as trabecular bone and periodontal ligament were significantly less visible and more variable among the systems in comparison with other anatomical structures (p=0.0001). Visibility of relatively large structures such as mandibular canal and mental foramen was satisfactory for all devices. The Accuitomo system was superior to MSCT and all other CBCT systems in depicting anatomical structures while MSCT was superior to all other CBCT systems in terms of reduced image noise. CONCLUSIONS: CBCT image quality is comparable or even superior to MSCT even though some variability exists among the different CBCT systems in depicting delicate structures. Considering the low radiation dose and high-resolution imaging, CBCT could be beneficial for dentomaxillofacial radiology.

Macro- and micro-anatomical, histological and computed tomography scan characterization of the nasopalatine canal.

AIM: To determine the human anatomic variability of the nasopalatine canal and determine its characteristics using an anatomical, histological and computed tomography (CT) scan evaluation. MATERIALS AND METHODS: Measurements for the canal characteristics were carried out on 163 dry human skulls and 120 upper jaw spiral CT scans, taken from patients for pre-operative planning purposes of implant placement in the incisor region. Furthermore, four human cadaver specimens were imaged using a high-resolution magnetic resonance imaging (HR-MRI) unit. Afterwards, these specimens were serially sectioned for histological examination to evaluate the nasopalatine canal region and its content. RESULTS: The nasopalatine canal anatomy showed a large variability in morphology and dimensions, with the canal branching in up to four canals at the level of the nose. The canal diameter was on average 3.3 mm (+/-0.9 mm SD), and typically enlarged by age and male gender (p<0.05). HR-MRI and histological sections enabled to identify the neurovascular structures within the canals. CONCLUSIONS: The large anatomic variations, the increased canal dimensions with age and the neurovascular canal content are all factors favouring a thorough three-dimensional planning before surgery, such as implant placement, of the anterior maxillary region.

Neurovascularization of the anterior jaw bones revisited using high-resolution magnetic resonance imaging.

The anterior jaw bones are often considered relatively safe surgical sites. Nonetheless, the increasing rate of surgical interventions in that area, such as oral implant placement and bone grafting, has highlighted the potential risks and has raised the reported complications. A careful documentation of all anatomic variations in anterior jaw bone neurovascularization has thus become necessary. The present report attempts to revisit jaw bone neurovascularization, addressing typical anatomic appearances and variations. We summarize the results of various microanatomical studies carried out by high-resolution magnetic resonance imaging (HR-MRI) of the human anterior jaw bones. These studies reveal that edentulous and dentate anterior jaws present significant variation in the occurrence of the mandibular incisive canal and genial spinal foramina, as well as the maxillary nasopalatine canal. All of these canal structures contain a neurovascular bundle, whose diameter may be large enough to cause clinically significant trauma. A careful presurgical radiographic analysis of the anterior jaw bones is therefore advised.