Polyetheretherketone bioactivity induced by farringtonite.

Polyetheretherketone (PEEK) is considered as an excellent biomaterial for bone grafting and connective tissue replacement. The clinical potential is, however, limited by its bioinertness, poor osteoconduction, and weak antibacterial activity. These disadvantages can be overcome by introducing suitable additives to produce mineral-polymer composites or coatings. In this work, a PEEK-based bioactive composite has been obtained by blending the polymer with magnesium phosphate (Mg3(PO4)2) particles in amounts ranging from 1 to 10 wt.% using the hot press technique. The obtained composite exhibited improved mechanical and physical properties, above the lower limits set for bone engineering applications. The tested grafts were found to not induce cytotoxicity. The presence of magnesium phosphate induced the mineralisation process with no adverse effects on the expression of the marker crucial for osteoblastic differentiation. The most promising results were observed in the grafts containing 1 wt.% of magnesium phosphate embedded within the PEEK matrix. The improved bioactivity of grafts, together with suitable physical-chemical and mechanical properties, indicate this composite as a promising orthopaedic implant material.

Markers of dental pulp stem cells in in vivo developmental context.

Teeth and their associated tissues contain several populations of mesenchymal stem cells, one of which is represented by dental pulp stem cells (DPSCs). These cells have mainly been characterised in vitro and numerous positive and negati ve markers for these cells have been suggested. To investigate the presence and localization of these molecules during development, forming dental pulp was examined using the mouse first mandibular molar as a model. The stages corresponding to postnatal (P) days 0, 7, 14, and 21 were investigated. The expression was monitored using customised PCR Arrays. Additionally, in situ localization of the key trio of markers (Cd73, Cd90, Cd105 coded by genes Nt5e, Thy1, Eng) was performed at prenatal and postnatal stages using immunohistochemistry. The expression panel of 24 genes assigned as in vitro markers of DPSCs or mesenchymal stem cells (MSCs) revealed their developmental dynamics during formation of dental pulp mesenchyme. Among the positive markers, Vcam1, Fgf2, Nes were identified as increasing and Cd44, Cd59b, Mcam, Alcam as decreasing between perinatal vs. postnatal stages towards adulthood. Within the panel of negative DPSC markers, Cd14, Itgb2, Ptprc displayed increased and Cd24a decreased levels at later stages of pulp formation. Within the key trio of markers, Nt5e did not show any significant expression difference within the investigated period. Thy1 displayed a strong decrease between P0 and P7 while Eng increased between these stages. In situ localization of Cd73, Cd90 and Cd105 showed them overlap in differentiated odontoblasts and in the sub-odontoblastic layer that is speculated to host odontoblast progenitors. The highly prevalent expression of particularly Cd73 and Cd90 opens the question of potential multiple functions of these molecules. The results from this study add to the in vitro based knowledge by showing dynamics in the expression of DPSC/MSC markers during dental pulp formation in an in vivo context and thus with respect to the natural environment important for commitment of stem cells.

Multiple epithelia are required to develop teeth deep inside the pharynx.

To explain the evolutionary origin of vertebrate teeth from odontodes, it has been proposed that competent epithelium spread into the oropharyngeal cavity via the mouth and other possible channels such as the gill slits [Huysseune et al., 2009, J. Anat. 214, 465-476]. Whether tooth formation deep inside the pharynx in extant vertebrates continues to require external epithelia has not been addressed so far. Using zebrafish we have previously demonstrated that cells derived from the periderm penetrate the oropharyngeal cavity via the mouth and via the endodermal pouches and connect to periderm-like cells that subsequently cover the entire endoderm-derived pharyngeal epithelium [Rosa et al., 2019, Sci. Rep. 9, 10082]. We now provide conclusive evidence that the epithelial component of pharyngeal teeth in zebrafish (the enamel organ) is derived from medial endoderm, as hitherto assumed based on position deep in the pharynx. Yet, dental morphogenesis starts only after the corresponding endodermal pouch (pouch 6) has made contact with the skin ectoderm, and only after periderm-like cells have covered the prospective tooth-forming endodermal epithelium. Manipulation of signaling pathways shown to adversely affect tooth development indicates they act downstream of these events. We demonstrate that pouch-ectoderm contact and the presence of a periderm-like layer are both required, but not sufficient, for tooth initiation in the pharynx. We conclude that the earliest interactions to generate pharyngeal teeth encompass those between different epithelial populations (skin ectoderm, endoderm, and periderm-like cells in zebrafish), in addition to the epithelial-mesenchymal interactions that govern the formation of all vertebrate teeth.

Large-Scale Automated Hollow-Fiber Bioreactor Expansion of Umbilical Cord-Derived Human Mesenchymal Stromal Cells for Neurological Disorders.

Neurodegenerative disorders present a broad group of neurological diseases and remain one of the greatest challenges and burdens to mankind. Maladies like amyotrophic lateral sclerosis, Alzheimer's disease, stroke or spinal cord injury commonly features astroglia involvement (astrogliosis) with signs of inflammation. Regenerative, paracrine and immunomodulatory properties of human mesenchymal stromal cells (hMSCs) could target the above components, thus opening new therapeutic possibilities for regenerative medicine. A special interest should be given to hMSCs derived from the umbilical cord (UC) tissue, due to their origin, properties and lack of ethical paradigms. The aim of this study was to establish standard operating and scale-up good manufacturing practice (GMP) protocols of UC-hMSCs isolation, characterization, expansion and comparison of cells' properties when harvested on T-flasks versus using a large-scale bioreactor system. Human UC-hMSCs, isolated by tissue explant culture technique from Wharton's jelly, were harvested after reaching 75% confluence and cultured using tissue culture flasks. Obtained UC-hMSCs prior/after the cryopreservation and after harvesting in a bioreactor, were fully characterized for "mesenchymness" immunomodulatory, tumorigenicity and genetic stability, senescence and cell-doubling properties, as well as gene expression features. Our study demonstrates an efficient and simple technique for large scale UC-hMSCs expansion. Harvesting of UC-hMSCs' using classic and large scale methods did not alter UC-hMSCs' senescence, genetic stability or in vitro tumorigenicity features. We observed comparable growth and immunomodulatory capacities of fresh, frozen and expanded UC-hMSCs. We found no difference in the ability to differentiate toward adipogenic, osteogenic and chondrogenic lineages between classic and large scale UC-hMSCs expansion methods. Both, methods enabled derivation of genetically stabile cells with typical mesenchymal features. Interestingly, we found significantly increased mRNA expression levels of neural growth factor (NGF) and downregulated insulin growth factor (IGF) in UC-hMSCs cultured in bioreactor, while IL4, IL6, IL8, TGFb and VEGF expression levels remained at the similar levels. A culturing of UC-hMSCs using a large-scale automated closed bioreactor expansion system under the GMP conditions does not alter basic "mesenchymal" features and quality of the cells. Our study has been designed to pave a road toward translation of basic research data known about human UC-MSCs for the future clinical testing in patients with neurological and immunocompromised disorders. An industrial manufacturing of UC-hMSCs next will undergo regulatory approval following advanced therapy medicinal products (ATMP) criteria prior to clinical application and approval to be used in patients.

Onset of calciotropic receptors during the initiation of mandibular/alveolar bone formation.

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.

Tuftelin and HIFs expression in osteogenesis.

Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.

Periderm invasion contributes to epithelial formation in the teleost pharynx.

The gnathostome pharyngeal cavity functions in food transport and respiration. In amniotes the mouth and nares are the only channels allowing direct contact between internal and external epithelia. In teleost fish, gill slits arise through opening of endodermal pouches and connect the pharynx to the exterior. Using transgenic zebrafish lines, cell tracing, live imaging and different markers, we investigated if pharyngeal openings enable epithelial invasion and how this modifies the pharyngeal epithelium. We conclude that in zebrafish the pharyngeal endoderm becomes overlain by cells with a peridermal phenotype. In a wave starting from pouch 2, peridermal cells from the outer skin layer invade the successive pouches until halfway their depth. Here the peridermal cells connect to a population of cells inside the pharyngeal cavity that express periderm markers, yet do not invade from outside. The latter population expands along the midline from anterior to posterior until the esophagus-gut boundary. Together, our results show a novel role for the periderm as an internal epithelium becomes adapted to function as an external surface.

Beyond the whole-mount phenotype: high-resolution imaging in fluorescence-based applications on zebrafish.

Zebrafish is now widely used in biomedical research as a model for human diseases, but the relevance of the model depends on a rigorous analysis of the phenotypes obtained. Many zebrafish disease models, experimental techniques and manipulations take advantage of fluorescent reporter molecules. However, phenotypic analysis often does not go beyond establishing overall distribution patterns of the fluorophore in whole-mount embryos or using vibratome or paraffin sections with poor preservation of tissue architecture and limited resolution. Obtaining high-resolution data of fluorescent signals at the cellular level from internal structures mostly depends on the availability of expensive imaging technology. Here, we propose a new and easily applicable protocol for embedding and sectioning of zebrafish embryos using in-house prepared glycol methacrylate (GMA) plastic that is suited for preservation of fluorescent signals (including photoactivatable fluorophores) without the need for antibodies. Four main approaches are described, all involving imaging fluorescent signals on semithin (3 µm or less) sections. These include sectioning transgenic animals, whole-mount immunostained embryos, cell tracking, as well as on-section enzyme histochemistry.

Fox proteins are modular competency factors for facial cartilage and tooth specification.

Facial form depends on the precise positioning of cartilage, bone, and tooth fields in the embryonic pharyngeal arches. How complex signaling information is integrated to specify these cell types remains a mystery. We find that modular expression of Forkhead domain transcription factors (Fox proteins) in the zebrafish face arises through integration of Hh, Fgf, Bmp, Edn1 and Jagged-Notch pathways. Whereas loss of C-class Fox proteins results in reduced upper facial cartilages, loss of F-class Fox proteins results in distal jaw truncations and absent midline cartilages and teeth. We show that Fox proteins are required for Sox9a to promote chondrogenic gene expression. Fox proteins are sufficient in neural crest-derived cells for cartilage development, and neural crest-specific misexpression of Fox proteins expands the cartilage domain but inhibits bone. These results support a modular role for Fox proteins in establishing the competency of progenitors to form cartilage and teeth in the face.

ARQ 087 inhibits FGFR signaling and rescues aberrant cell proliferation and differentiation in experimental models of craniosynostoses and chondrodysplasias caused by activating mutations in FGFR1, FGFR2 and FGFR3.

Tyrosine kinase inhibitors are being developed for therapy of malignancies caused by oncogenic FGFR signaling but little is known about their effect in congenital chondrodysplasias or craniosynostoses that associate with activating FGFR mutations. Here, we investigated the effects of novel FGFR inhibitor, ARQ 087, in experimental models of aberrant FGFR3 signaling in cartilage. In cultured chondrocytes, ARQ 087 efficiently rescued all major effects of pathological FGFR3 activation, i.e. inhibition of chondrocyte proliferation, loss of extracellular matrix and induction of premature senescence. In ex vivo tibia organ cultures, ARQ 087 restored normal growth plate architecture and eliminated the suppressing FGFR3 effect on chondrocyte hypertrophic differentiation, suggesting that it targets the FGFR3 pathway specifically, i.e. without interference with other pro-growth pathways. Moreover, ARQ 087 inhibited activity of FGFR1 and FGFR2 mutants associated with Pfeiffer, Apert and Beare-Stevenson craniosynostoses, and rescued FGFR-driven excessive osteogenic differentiation in mouse mesenchymal micromass cultures or in ex vivo calvarial organ cultures. Our data warrant further development of ARQ 087 for clinical use in skeletal disorders caused by activating FGFR mutations.

Osteogenic Profile of Mesenchymal Cell Populations Contributing to Alveolar Bone Formation.

Teeth develop within the surrounding periodontal tissues, involving the alveolar bone, periodontal ligament and cementum. The alveolar bone originates through the process of intramembranous ossification involving mesenchymal cells from the tooth germ. As most available data are related to endochondral ossification, we examined the molecular background of alveolar bone development. We investigated the osteogenic profile of mesenchymal cells dissected from mouse mandible slices at the stage of early alveolar bone formation. Relative monitoring of gene expression was undertaken using PCR Arrays; this included the profiles of 84 genes associated with osteogenesis. To examine the tooth-bone interface, stages with detectable changes in bone remodelling during development (E13.0, E14.0 and E15.0) were chosen and compared with each other. These results showed a statistically significant increase in the expression of the genes Fgf3, Ctsk, Icam-1, Mmp9, Itga3 and Tuft1, and of a wide range of collagens (Col1a2, Col3a1, Col7a1, Col12a1, Col14a1). Decreased expression was detected in the case of Col2a1, Sox9, Smad2 and Vegfb. To confirm these changes in gene expression, immunofluorescence analyses of Mmp9 and Sox9 proteins were performed in situ. Our research has identified several candidate genes that may be crucial for the initiation of alveolar bone formation and is the basis for further functional studies.

Fibroblast growth factor and canonical WNT/ß-catenin signaling cooperate in suppression of chondrocyte differentiation in experimental models of FGFR signaling in cartilage.

Aberrant fibroblast growth factor (FGF) signaling disturbs chondrocyte differentiation in skeletal dysplasia, but the mechanisms underlying this process remain unclear. Recently, FGF was found to activate canonical WNT/ß-catenin pathway in chondrocytes via Erk MAP kinase-mediated phosphorylation of WNT co-receptor Lrp6. Here, we explore the cellular consequences of such a signaling interaction. WNT enhanced the FGF-mediated suppression of chondrocyte differentiation in mouse limb bud micromass and limb organ cultures, leading to inhibition of cartilage nodule formation in micromass cultures, and suppression of growth in cultured limbs. Simultaneous activation of the FGF and WNT/ß-catenin pathways resulted in loss of chondrocyte extracellular matrix, expression of genes typical for mineralized tissues and alteration of cellular shape. WNT enhanced the FGF-mediated downregulation of chondrocyte proteoglycan and collagen extracellular matrix via inhibition of matrix synthesis and induction of proteinases involved in matrix degradation. Expression of genes regulating RhoA GTPase pathway was induced by FGF in cooperation with WNT, and inhibition of the RhoA signaling rescued the FGF/WNT-mediated changes in chondrocyte cellular shape. Our results suggest that aberrant FGF signaling cooperates with WNT/ß-catenin in suppression of chondrocyte differentiation.

Distribution of BMP6 in the alveolar bone during mouse mandibular molar eruption.

Eruption requires synchrony of the tooth with the surrounding tissues, particularly the bone. One important step during eruption is remodelling of the alveolar bone at the base of the tooth and along the roots. Expression of BMP6 was reported to be increased in the basal half of the dental follicle prior to eruption and inhibition of BMP6 affected bone formation at the base of the alveolar crypt. The aim of this study was to further investigate BMP6 protein in relation to tooth eruption and the corresponding bone remodelling using temporospatial correlations of BMP6 localization with morphogenetic events (proliferation, differentiation, apoptosis and bone apposition/resorption), other BMPs (BMP2 and BMP7) and three-dimensional images of tooth-bone development. BMP6 expression pattern was mapped in the mandibular molar teeth and related structures around eruption. Localization of BMP6 dominated in osteoblasts, in regions of bone formation within the alveolar crypt. These findings positively correlated with proliferation at the tooth base region, osteocalcin expression in the osteoblasts/osteocytes and BMP2 and BMP7 presence in the alveolar bone surrounding the tooth. Osteoclast activity and apoptotic elimination in the root region gradually decreased before eruption and totally ceased at eruption stages. Generally, BMP6 positively correlated with BMP2, BMP7 and osteocalcin-positive osteoblasts, and areas of bone remodelling. Moreover, BMP6 was found in the periodontium and cementoblasts. BMP6 expression in the alveolar bone accompanied tooth eruption. Notably, the expression pattern of BMP6 in the bone did not differ around individual molar teeth at the same stage of development. The expression of BMP6 in periodontal ligaments may contribute to interaction between the tooth and bone during the eruption and anchoring process.