Early Stages Of Bone Formation Research Articles

Would three BMPs at low concentration be better than one at high concentration? An experimental study with rat osteoprogenitor cells.

Bone morphogenetic proteins (BMPs) are used in clinical practice for stimulation of bone formation, but often evoke serious complications. Recent studies demonstrated that BMPs involved in early stages of bone formation are species specific. In cattle dominate BMP7, growth differentiation factor 5 (GDF5) and NEL-like protein 1 (NELL1) while in rats BMP2, BMP5 and BMP6. The purpose of the study was to compare the action of the species specific BMPs on the osteoprogenitor cells. Thus, rat osteoprogenitor cells were exposed to one BMP in a high dose and three of them at 1/3 of the former. Isolated rat osteoprogenitor cells were treated in culture with different concentrations of BMP2, BMP5 and BMP6 or with lower concentration of combinations of these cytokines. Activity of alkaline phosphatase, calcium deposition and mRNA level for transcription factor SP7 (osterix) and tissue non-specific alkaline phosphatase (TNAP) served as indicators of BMPs effect. BMPs stimulated all studied parameters in comparison with control cultures, but no statistically significant differences were observed between the action of a large dose of one cytokine and a combination of cytokines given at lower concentrations. Three BMPs used in a low dose exert similar effect as the one used at high dose. Since the BMPs stimulate different receptors and activate different signaling pathways the use of the mixture of properly chosen BMPs at low concentration may give better results than the single one at high concentration and may avoid untoward effects.

In vivo imaging of bone collagen dynamics in zebrafish

Type I collagen plays a pivotal role in shaping bone morphology and determining its physical properties by serving as a template for ossification. Nevertheless, the mechanisms underlying bone collagen formation, particularly the principles governing its orientation, remain unknown owing to the lack of a method that enables continuous in vivo observations. To address this challenge, we constructed a method to visualize bone collagen by tagging with green fluorescent protein (GFP) in zebrafish and observed the interactions between osteoblasts and collagen fibers during bone formation in vivo. When collagen type I alpha 2 chain (Col1a2)-GFP was expressed under the control of the osteoblast-specific promoters osx or osc in zebrafish, bone collagen was observed clearly enough to identify its localization, whereas collagen from other organs was not. Therefore, we determined that this method was of sufficient quality for the detailed in vivo observation of bone collagen. Next, bone collagen in the scales, fin rays, and opercular bones of zebrafish was observed in detail, when bone formation is more active. High-magnification imaging showed that Col1a2-GFP can visualize collagen sufficiently to analyze the collagen fiber orientation and microstructure of bones.Furthermore, by simultaneously observation of bone collagen and osteoblasts, we successfully observed dynamic changes in the morphology and position of osteoblasts from the early stages of bone formation. It was also found that the localization pattern and orientation of bone collagen significantly differed depending on the choice of the expression promoter. Both promoters (osx and osc) used in this study are osteoblast-specific, but their Col1a2-GFP localizing regions within the bone were exclusive, with osx region localizing mainly to the outer edge of the bone and osc region localizing to the central area of the bone. This suggests the existence of distinct osteoblast subpopulations with different gene expression profiles, each of which may play a unique role in osteogenesis.These findings would contribute to a better understanding of the mechanisms governing bone collagen formation by osteoblasts.

Bio-inspired liquid crystal gel induction via nano-hydroxyapatite mesogens: Viscoelastic and hemostasis regulation under bone remodeling pH and temperature control

In this work, a liquid crystal mesophase as a potential bone-repairing booster has been induced from a polyacrylic acid (PAAc) gel loaded with synthetic hydroxyapatite nanoparticles (nHA) under physiological conditions of pH and temperature that resemble the early stages of bone formation. Dynamic processes at the microscale in biological systems require a material capable of behaving following a specific order and fluency. Besides, they must fulfill conditions such as biocompatibility and good mechanical properties. Our study aimed to obtain new data about the safety, hemostasis, biocompatibility (activated partial thromboplastin time (APTT) test, prothrombin time (PT) test, lactate dehydrogenase (LDH) test, red blood cell morphology observation, hemolysis percentage) and physicochemical/mechanical behavior (Attenuated Total Reflection (ATR), Infrared spectroscopy and rheological assays) of nanoparticles, and materials built with them. Both systems have shown biocompatibility with red blood cells and adequate flow properties, demonstrating promising qualities for applicability. The formation of lyotropic liquid crystals at physiological conditions was evaluated with crossed polaroids microscope with a temperature control plate. The formation of these structures under bone remodeling conditions shows the potential application of PAAc-nHA materials. In literature, the study of materials applied as bone fillers is mainly focused on mechanical and osteoblasts compatibility assays. However, the hematological effect, consequences on the coagulation cascade, the integrity of cell membranes, and hemolysis percentage have not been addressed thoroughly. The strength of our work is focused on considering these aspects when tridimensional arrangements essential for bone formation are formed.

The collagen receptor, discoidin domain receptor 2, functions in Gli1-positive skeletal progenitors and chondrocytes to control bone development

Discoidin Domain Receptor 2 (DDR2) is a collagen-activated receptor kinase that, together with integrins, is required for cells to respond to the extracellular matrix. Ddr2 loss-of-function mutations in humans and mice cause severe defects in skeletal growth and development. However, the cellular functions of Ddr2 in bone are not understood. Expression and lineage analysis showed selective expression of Ddr2 at early stages of bone formation in the resting zone and proliferating chondrocytes and periosteum. Consistent with these findings, Ddr2+ cells could differentiate into hypertrophic chondrocytes, osteoblasts, and osteocytes and showed a high degree of colocalization with the skeletal progenitor marker, Gli1. A conditional deletion approach showed a requirement for Ddr2 in Gli1-positive skeletal progenitors and chondrocytes but not mature osteoblasts. Furthermore, Ddr2 knockout in limb bud chondroprogenitors or purified marrow-derived skeletal progenitors inhibited chondrogenic or osteogenic differentiation, respectively. This work establishes a cell-autonomous function for Ddr2 in skeletal progenitors and cartilage and emphasizes the critical role of this collagen receptor in bone development.

Human Periodontal Ligament Stem Cells Response to Titanium Implant Surface: Extracellular Matrix Deposition.

Simple SummaryThe extracellular matrix (ECM) is a fundamental component of tissues and organs, providing the structural and non-structural support that leads to the bone osseointegration. Understanding the mechanisms of ECM components modulation is essential for developing novel strategies for tissue engineering and regenerative medicine and in particular for the dental implant surface design. The release of ECM components by cells is the first step that leads to the early stage of bone formation. The present work is focused on the modulation of ECM components expression by human periodontal ligament stem cells (hPDLSCs) cultured on two different dental implant surfaces, sandblasted (CTRL) and dual acid-etched (TEST). The effects were evaluated by means of the morphological observations and protein and gene expression. The results demonstrated that the rough surface of titanium dental implant can enhance the expression of ECM molecules and osteogenic markers in hPDLSCs. The surface topography is of vital importance as it regulates cell response. It can be considered one of the main factors that influence the success of a dental implant. The influence of surface topography on osseointegration could lead to short healing times and a better quality of oral rehabilitation and patient life.The major challenge for dentistry is to provide the patient an oral rehabilitation to maintain healthy bone conditions in order to reduce the time for loading protocols. Advancement in implant surface design is necessary to favour and promote the osseointegration process. The surface features of titanium dental implant can promote a relevant influence on the morphology and differentiation ability of mesenchymal stem cells, induction of the osteoblastic genes expression and the release of extracellular matrix (ECM) components. The present study aimed at evaluating the in vitro effects of two different dental implants with titanium surfaces, TEST and CTRL, to culture the human periodontal ligament stem cells (hPDLSCs). Expression of ECM components such as Vimentin, Fibronectin, N-cadherin, Laminin, Focal Adhesion Kinase (FAK) and Integrin beta-1 (ITGB1), and the osteogenic related markers, as runt related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP), were investigated. Human PDLSCs cultured on the TEST implant surface demonstrated a better cell adhesion capability as observed by Scanning Electron Microscopy (SEM) and immunofluorescence analysis. Moreover, immunofluorescence and Western blot experiments showed an over expression of Fibronectin, Laminin, N-cadherin and RUNX2 in hPDLSCs seeded on TEST implant surface. The gene expression study by RT-PCR validated the results obtained in protein assays and exhibited the expression of RUNX2, ALP, Vimentin (VIM), Fibronectin (FN1), N-cadherin (CDH2), Laminin (LAMB1), FAK and ITGB1 in hPDLSCs seeded on TEST surface compared to the CTRL dental implant surface. Understanding the mechanisms of ECM components release and its regulation are essential for developing novel strategies in tissue engineering and regenerative medicine. Our results demonstrated that the impact of treated surfaces of titanium dental implants might increase and accelerate the ECM apposition and provide the starting point to initiate the osseointegration process.

Muscular loading affects the 3D structure of both the mineralized rudiment and growth plate at early stages of bone formation.

Fetal immobilization affects skeletal development and can lead to severe malformations. Still, how mechanical load affects embryonic bone formation is not fully elucidated. This study combines mechanobiology, image analysis and developmental biology, to investigate the structural effects of muscular loading on embryonic long bones. We present a novel approach involving a semi-automatic workflow, to study the spatial and temporal evolutions of both hard and soft tissues in 3D without any contrast agent at micrometrical resolution. Using high-resolution phase-contrast-enhanced X-ray synchrotron microtomography, we compare the humeri of Splotch-delayed embryonic mice lacking skeletal muscles with healthy littermates. The effects of skeletal muscles on bone formation was studied from the first stages of mineral deposition (Theiler Stages 23 and 24) to just before birth (Theiler Stage 27). The results show that muscle activity affects both growth plate and mineralized regions, especially during early embryonic development. When skeletal muscles were absent, there was reduced mineralization, altered tuberosity size and location, and, at early embryonic stages, decreased chondrocyte density, size and elongation compared to littermate controls. The proposed workflow enhances our understanding of mechanobiology of early bone formation and could be implemented for the study of other complex biological tissues.

Enhanced Osseointegration by the Hierarchical Micro-Nano Topography on Selective Laser Melting Ti-6Al-4V Dental Implants.

Currently, selective laser melting (SLM) has been thriving in implant dentistry for on-demand fabricating dental implants. Based on the coarse microtopography of SLM titanium surfaces, constructing nanostructure to form the hierarchical micro-nano topography is effective in enhancing osseointegration. Given that current nanomodification techniques of SLM implants, such as anodization and hydrothermal treatment, are facing the inadequacy in costly specific apparatus and reagents, there has been no recognized nanomodified SLM dental implants. The present study aimed to construct hierarchical micro-nano topography on self-made SLM dental implants by a simple and safe inorganic chemical oxidation, and to evaluate its contribution on osteoblastic cells bioactivity and osseointegration. The surface chemical and physical parameters were characterized by FE-SEM, EDS, profilometer, AFM, and contact angle meter. The alteration on bioactivity of MG-63 human osteoblastic cells were detected by qRT-PCR. Then the osseointegration was assessed by implanting implants on the femur condyle of New Zealand Rabbits. The hierarchical micro-nano topography was constituted by the microrough surface of SLM implants and nanoneedles (diameter: 20∼50 nm, height: 150∼250 nm), after nanomodifying SLM implants in 30% hydrogen peroxide and 30% hydrochloride acid (volume ratio 1:2.5) at room temperature for 36 h. Low chemical impurities content and high hydrophilicity were observed in the nanomodified group. Cell experiments on the nanomodified group showed higher expression of mitophagy related gene (PINK1, PARKIN, LC3B, and LAMP1) at 5 days and higher expression of osteogenesis related gene (Runx2 and OCN) at 14 days. In the early stage of bone formation, the nanomodified SLM implants demonstrated higher bone-to-implant contact. Intriguingly, the initial bone-to-implant contact of nanomodified SLM implants consisted of more mineralized bone with less immature osteoid. After the cessation of bone formation, the bone-to-implant contact of nanomodified SLM implants was equal to untreated SLM implants and marketable TixOs implants. The overall findings indicated that the inorganic chemical oxidized hierarchical micro-nano topography could enhance the bioactivity of osteoblastic cells, and consequently promote the peri-implant bone formation and mineralization of SLM dental implants. This study sheds some light on improvements in additive manufactured dental implants.

Lineage tracing of cells expressing the ciliary gene IFT140 during bone development.

Primary cilia influence cell function and tissue development. Ciliary signaling is mediated by two intraflagellar transport (IFT) protein complexes, IFT-A and IFT-B. The IFT-A complex is responsible for retrograde transport, and IFT140 is a core protein in the A complex. Mutations in IFT140 cause a variety of skeletal disorders. However, the expression and role of IFT140 during bone development remain unclear. In this study, to further explore the potential role of IFT140 in osteogenesis, we used cell lineage tracing and conditional knockout to analyze the distribution and function of IFT140-positive cells during bone formation. In newborn Ift140-creER; R26RtdTomato mice, IFT140-positive cells were mainly located in the medullary cavity and then migrated to and differentiated on the surface of trabecular and cortical bone. In contrast, the number of IFT140-positive cells significantly decreased in the adult stage, and these cells were only located in the bone marrow cavity for a short time. In Osx-cre; Ift140flox/flox mice, the loss of IFT140 in preosteoblasts caused bone loss in the trabecular bone area at 10 weeks. The results revealed that IFT140-positive cells mainly contribute to the early stage of bone formation.

Periostin Mediates Oestrogen-Induced Osteogenic Differentiation of Bone Marrow Stromal Cells in Ovariectomised Rats.

Osteoporosis is a metabolic disease that results in the progressive loss of bone mass, which, in postmenopausal women, is related to oestrogen deficiency. Periostin (POSTN) plays a key role in the early stages of bone formation. However, whether POSTN participates in oestradiol regulation of osteogenic differentiation of bone marrow stromal cells (BMSCs) from ovariectomised (OVX) rats remains unclear. In vivo, using microcomputed tomography (micro-CT), immunohistochemistry, and dynamic analysis of femurs, we found that 17β-E2 promotes bone formation and POSTN expression at the endosteal surface. In vitro, 17β-E2 upregulated POSTN expression in OVX-BMSCs. POSTN overexpression activated the Wnt/β-catenin signalling pathway and enhanced osteogenic differentiation of OVX-BMSCs. Furthermore, knockdown of Postn blocks the involvement of 17β-E2 in the osteogenic differentiation of OVX-BMSCs. Collectively, our study indicated the role of POSTN in the osteogenesis and stemness of OVX-BMSCs and proves that 17β-E2 reduces osteoporosis and promotes osteogenesis through the POSTN-Wnt/β-catenin pathway. POSTN could, therefore, be a novel target gene for anti-osteoporosis therapies.

Biomimetic Composite Scaffold With Phosphoserine Signaling for Bone Tissue Engineering Application.

In guided bone tissue engineering, successful ingrowth of MSCs depends primarily on the nature of the scaffold. It is well-known that only seconds after implantation, biomaterials are coated by a layer of adsorbed proteins/peptides which modulates the subsequent cell/scaffold interactions, especially at early times after implantation. In this work, nanohydroxyapatite and collagen based composite materials (Coll/nanoHA) were modified with phosphorylated amino acid (O-phospho-L-serine–OPS) to mimic bone tissue, and induce cell differentiation. The choice for this phosphorylated amino acid is due to the fact that osteopontin is a serine-rich glycol-phosphoprotein and has been associated to the early stages of bone formation, and regeneration. Several concentrations of OPS were added to the Coll/nanoHA scaffold and physico-chemical, mechanical, and in vitro cell behavior were evaluated. Afterwards, the composite scaffold with stronger mechanical and best cellular behavior was tested in vivo, with or without previous in vitro culture of human MSC's (bone tissue engineering). The OPS signaling of the biocomposite scaffolds showed similar cellular adhesion and proliferation, but higher ALP enzyme activity (HBMSC). In vivo bone ectopic formation studies allowed for a thorough evaluation of the materials for MSC's osteogenic differentiation. The OPS-scaffolds results showed that the material could modulated mesenchymal cells behavior in favor of osteogenic differentiation into late osteoblasts that gave raised to their ECM with human bone proteins (osteopontin) and calcium deposits. Finally, OPS-modified scaffolds enhanced cell survival, engraftment, migration, and spatial distribution within the 3D matrix that could be used as a cell-loaded scaffold for tissue engineering applications and accelerate bone regeneration processes.

Engineered mosaic protein polymers; a simple route to multifunctional biomaterials

BackgroundEngineered living materials (ELMs) are an exciting new frontier, where living organisms create highly functional materials. In particular, protein ELMs have the advantage that their properties can be manipulated via simple molecular biology. Caf1 is a protein ELM that is especially attractive as a biomaterial on account of its unique combination of properties: bacterial cells export it as a massive, modular, non-covalent polymer which is resistant to thermal and chemical degradation and free from animal material. Moreover, it is biologically inert, allowing the bioactivity of each 15 kDa monomeric Caf1 subunit to be specifically engineered by mutagenesis and co-expressed in the same Escherichia coli cell to produce a mixture of bioactive Caf1 subunits.ResultsHere, we show by gel electrophoresis and transmission electron microscopy that the bacterial cells combine these subunits into true mosaic heteropolymers. By combining two separate bioactive motifs in a single mosaic polymer we demonstrate its utility by stimulating the early stages of bone formation by primary human bone marrow stromal cells. Finally, using a synthetic biology approach, we engineer a mosaic of three components, demonstrating that Caf1 complexity depends solely upon the variety of monomers available.ConclusionsThese results demonstrate the utility of engineered Caf1 mosaic polymers as a simple route towards the production of multifunctional biomaterials that will be useful in biomedical applications such as 3D tissue culture and wound healing. Additionally, in situ Caf1 producing cells could create complex bacterial communities for biotechnology.Graphical abstract

Quantitative in situ time-series evaluation of osteoblastic collagen synthesis under cyclic strain using second-harmonic-generation microscopy.

.The aim of this study is to evaluate the osteoblastic collagen synthesis under mechanical stimulation using second-harmonic-generation (SHG) microscopy. We apply SHG microscopy to monitor the collagen fibers synthesized by osteoblast-like cells (MC3T3-E1) without the need for fixation and staining. To quantitatively evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis, we compare SHG images of osteoblast-synthesized collagen fibers with and without a cyclic stretch stimulus applied using a lab-made stretching device. We acquire SHG images every 7 days for 3 weeks at different stimulus conditions (5 min/day and 3 h/day with a strain magnitude of 5% and a frequency of 0.5 Hz). Image analysis of the average SHG intensity indicates that the amount of osteoblastic collagen synthesis is significantly enhanced by the cyclic stretch compared with the nonstretched condition, while there is no significant difference between the two mechanical stimulation conditions. Furthermore, the maturity of the collagen fibers in the early stage of bone formation is not affected by the mechanical stimulation. The results can be used in bone regenerative medicine to apply feedback control of collagen synthesis by artificial stimulation.

Breath Figures decorated silicon oxinitride ceramic surfaces with controlled Si ions release for enhanced osteoinduction.

Bioactive coatings are usually applied to bone and dental prostheses to enhance the integration and their stability in the bone. Recently, silicon (Si) oxynitride ceramics have been demonstrated to possess osteoconductive properties due to the release of Si ions, particularly important in the early stage of bone formation. In addition, the pattern of the bone contacting surface has been reported to affect cells' differentiation and metabolic activity. In this work, we propose the Breath Figure (BF) process combined with a pyrolysis step, starting from a photo-crosslinkable alkoxy silicone precursor, as a method to realize bioactive patterned coating on metal bone and dental prostheses. Four different surface patterned coatings were applied to Ti4Al6V disks starting from solutions with different precursor concentrations. Morphology, chemical composition, and Si ions' release were evaluated and compared. Moreover, all samples underwent to biological in vitro testing with human mesenchymal stem cells (hMSCs) in comparison with the uncoated titanium alloy. The results indicated that the Si released from the coatings determined an increase in the cellular activity with the BF pattern influencing the hMSCs' initial adhesion and proliferation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1284-1294, 2019.

E3-ubiquitin ligase NEDD4 enhances bone formation by removing TGFβ1-induced pSMAD1 in immature osteoblast.

Neural precursor cell expressed developmentally downregulated protein 4 (NEDD4) is an E3 ubiquitin ligase that regulates animal growth and development. To investigate the role of NEDD4 in skeletogenesis in vivo, we established immature osteoblast-specific 2.3-kb Collagen Type I Alpha 1 chain (Col1α1) promoter-driven Nedd4 transgenic (Nedd4-TG, Col1α1-Nedd4Tg/+) mice and conditional knockout (Nedd4-cKO, Col1α1-Cre;Nedd4fl/fl) mice. The Nedd4-TG mice displayed enhanced bone mass accrual and upregulated gene expression of osteogenic markers in bone. In addition, bone formation was decreased in the Nedd4-cKO mice compared to that in their littermates. The proliferation of primary osteoblasts isolated from calvaria and the number and surface area of tibial osteoblasts were higher in the Nedd4-TG mice than those in their littermates. Throughout the osteoblast differentiation, the expression of Nedd4 and Tgfb1 were high at early stage of osteoblast maturation, but decreased at the later stage when Bmp2 expression level is high. TGFβ1 signaling was consolidated by degradation of pSMAD1, which was transiently induced by TGFβ1, in NEDD4-overexpressing osteoblasts. Furthermore, pERK1/2 signaling was enhanced in osteoblast from TG mice than those in their littermates. These results suggest that NEDD4 enhances osteoblast proliferation by removing pSMAD1 activated by TGFβ1, and potentiating pSMAD2 and pERK1/2 pathways at early stage of bone formation.

Effect of Laser Bio-Stimulation on Mandibular Distraction Osteogenesis: An Experimental Study

The aim of this study was to evaluate the osseous response to laser bio-stimulation clinically and histologically during distraction osteogenesis (DO) induced in the mandibles of mongrel dogs. Thirty dogs were divided into 3 groups of 10 (5 with and 5 without laser treatment) according to sacrifice periods (2, 4, and 8weeks after distraction). DO was performed between the mandibular second and third premolars using an internal linear distractor. After a 7-day latency period, the distractor was activated at the rate of 1mm per day for 10days followed by a consolidation period during which the right mandibular side was irradiated with a diode laser (wavelength [λ], 970nm; power, 2W; spot size, 320μm; total energy [E], 840J), whereas the control group was not irradiated, after distraction. Histologic specimens were prepared and histomorphometric analysis of specimens was performed. Clinical examinations showed that the low-intensity laser diode had a pronounced effect on the quality and quantity of newly formed bone in the DO regenerate in the laser groups compared with the control groups. Histopathologic sections from laser groups I, II, and III displayed the bio-stimulatory effect of laser on new bone through an increased rate of osteoblast proliferation and differentiation, an accelerated rate of intramembranous ossification, and increased neoangiogenesis compared with the control groups. Moreover, the histomorphometric results showed that mean bone trabecular size, bone trabecular total area, and bony area fraction of the regenerate were larger and statistically significant (P<.05) especially in laser groups I and II (early stages of bone formation) compared with the control groups. The low-level diode laser had a positive role as a potential bio-stimulator and local inducer in enhancing bone formation during DO and resulted in early stability of the bone regenerate, a shorter total treatment time, and improved new bone quality and quantity.

Biofabrication of a co-culture system in an osteoid-like hydrogel matrix

Biofabrication aims to develop functional, biological constructs using automated processes (additive manufacturing, AM) involving different cell types and biomaterials (Groll et al 2016 Biofabrication 13001 1–6). As bone tissue is based on the crosstalk between osteoblasts and osteoclasts at least, evaluating cell–cell and cell–material interactions is of interest to understand bone remodeling. There is increasing interest in the role of osteoclasts not only considering bone resorption, but also their influence on the proliferation, migration and differentiation of osteoblasts. Osteoid-like, non-mineralized matrix is used here for the 3D cultivation of osteoblast and osteoclast progenitor cells to evaluate interactions in an early stage of bone formation. The AM technology bioplotting was used to tailor a 3D environment with defined properties. These results could be helpful to transfer this approach to the fabrication of bone tissue in regenerative medicine approaches. Gelatin is derived from collagen, which is the main phase of osteoid. Oxidized alginate–gelatin crosslinked hydrogel was used to immobilize osteoblastic (ST2) and osteoclastic (RAW) progenitor cells. Cell viability and number, the expression of different proteins like alkaline phosphatase (ALP), osteopontin (OPN) and tartrate resistant acid phosphatase (TRAP) were investigated. Release of vascular endothelial growth factor (VEGF) by the immobilized cells was analyzed. Microscopy techniques were used to evaluate cell morphology during an incubation period of 21 days. The biofabrication process was compatible with the cells. Cells migrated, proliferated and expressed their specific proteins indicating cell differentiation. The co-culture showed increased OPN concentration, which is a major protein of the osteoid involved in the mineralization process. TRAP activity was increased compared to single culture. ST2 single culture showed higher ALP activity compared to the co-culture. VEGF concentration of the co-culture was strongly increased. The results indicate the importance of using co-cultures to fabricate bone tissue by biofabrication. Especially the influence of the osteoblast/osteoclast crosstalk, in an early stage of bone formation, is shown here, using a 3D hydrogel based cell culture model created by biofabrication.

Role of Cortico-Cancellous Heterologous Bone in Human Periodontal Ligament Stem Cell Xeno-Free Culture Studied by Synchrotron Radiation Phase-Contrast Microtomography.

This study was designed to quantitatively demonstrate via three-dimensional (3D) images, through the Synchrotron Radiation Phase-Contrast Microtomography (SR-PhC-MicroCT), the osteoinductive properties of a cortico-cancellous scaffold (Osteobiol Dual Block—DB) cultured with human Periodontal Ligament Stem Cells (hPDLSCs) in xeno-free media. In vitro cultures of hPDLSCs, obtained from alveolar crest and horizontal fibers of the periodontal ligament, were seeded onto DB scaffolds and cultured in xeno-free media for three weeks. 3D images were obtained by SR-PhC-microCT after one and three weeks from culture beginning. MicroCT data were successively processed with a phase-retrieval algorithm based on the Transport of Intensity Equation (TIE). The chosen experimental method, previously demonstratively applied for the 3D characterization of the same constructs in not xeno-free media, quantitatively monitored also in this case the early stages of bone formation in basal and differentiating conditions. Interestingly, it quantitatively showed in the xeno-free environment a significant acceleration of the mineralization process, regardless of the culture (basal/differentiating) medium. This work showed in 3D that the DB guides the osteogenic differentiation of hPDLSCs in xeno-free cultures, in agreement with 2D observations and functional studies previously performed by some of the authors. Indeed, here we fully proved in 3D that expanded hPDLSCs, using xeno-free media formulation, not only provide the basis for Good Manufacturing Practice (preserving the stem cells’ morphological features and their ability to differentiate into mesenchymal lineage) but have to be considered, combined to DB scaffolds, as interesting candidates for potential clinical use in new custom made tissue-engineered constructs.

In vitro effects of mechanical stimulation and photobiomodulation on osteoblastic cell function: A proof of concept study

BackgroundThe effect of mechanical stimulation and photobiomodulation on tissue defect restoration has recently been investigated in various fields of tissue engineering such as acceleration of healing process and bone calcification. Mechanical stimulation generates shear stress on bone cells resulting in promotion of bone formation whereas photobiomodulation regulates inflammation, decreases pain, accelerates cell proliferation and enhances healing. MethodsMC3T3-E1 cells were cultured in 3 dimensional collagen scaffolds. Cells were daily stimulated by either mechanical loading of 3 Hz sinusoidal with 3000 μstrain vibration, or photobiomodulation using LED with 3 J/cm2 fluency or combination of both. The calcifications of 3D tissue-engineered bones were examined by non-destructive monitoring device every day for 42 days. ResultsThe 3D tissue-engineered bones that exposed to mechanical alone or combined stimulation exhibited early calcification, higher calcium content and bulk density comparing to control and light stimulation alone. Furthermore, the mRNA expression level of bone formation related genes such as RUNX2, ALP, osteopontin and osteocalcin were examined 7 days after stimulations. We showed the potential upregulation of ALP gene after mechanical stimulation alone or combined with light treatment. On day 28 Von Kossa stain revealed higher calcium deposition and increased cell migration to the deeper zone of 3D tissue-engineered bones. ConclusionWe suggested that the mechanical treatment alone and combination with light treatment could accelerate the calcification of 3D tissue-engineered bone possibly through up-regulation of ALP gene during early stage of bone formation.

Synchrotron-based XRD from rat bone of different age groups

Synchrotron-based XRD spectra from rat bone of different age groups (w, 56 w and 78w), lumber vertebra at early stages of bone formation, Calcium hydroxyapatite (HAp) [Ca10(PO4)6(OH)2] bone fill with varying composition (60% and 70%) and bone cream (35–48%), has been acquired with 15keV synchrotron X-rays. Experiments were performed at Desy, Hamburg, Germany, utilizing the Resonant and Diffraction beamline (P9), with 15keV X-rays (λ=0.82666 A0). Diffraction data were quantitatively analyzed using the Rietveld refinement approach, which allowed us to characterize the structure of these samples in their early stages. Hydroxyapatite, received considerable attention in medical and materials sciences, since these materials are the hard tissues, such as bone and teeth. Higher bioactivity of these samples gained reasonable interest for biological application and for bone tissue repair in oral surgery and orthopedics. The results obtained from these samples, such as phase data, crystalline size of the phases, as well as the degree of crystallinity, confirm the apatite family crystallizing in a hexagonal system, space group P63/m with the lattice parameters of a=9.4328Å and c=6.8842Å (JCPDS card #09-0432). Synchrotron-based XRD patterns are relatively sharp and well resolved and can be attributed to the hexagonal crystal form of hydroxyapatite. All the samples were examined with scanning electron microscope at an accelerating voltage of 15kV. The presence of large globules of different sizes is observed, in small age groups of the rat bone (8w) and lumber vertebra (LV), as distinguished from, large age groups (56 and 78w) in all samples with different magnification, reflects an amorphous phase without significant traces of crystalline phases. Scanning electron microscopy (SEM) was used to characterize the morphology and crystalline properties of Hap, for all the samples, from 2 to 100μm resolution.

Hydrophilic surface of Ti6Al4V-ELI alloy improves the early bone apposition of sheep tibia.

The aim of this preclinical study was to compare histologically and histomorphometrically both sandblasted/acid-etched implant surfaces with or without maintained in an isotonic solution of 0.9% sodium chloride in early stages of osseointegration. Both implant surfaces were composed of a titanium/aluminum/vanadium alloy (Ti6Al4V-ELI), but they had different surface chemistries: sandblasted/acid-etched titanium surface (FN) or sandblasted/acid-etched surface maintained in an isotonic solution of 0.9% sodium chloride (FA). The surface morphology, topography and chemistry were evaluated by scanning electron microscopy (SEM), confocal microscopy (CM) and X-ray photoelectron spectroscopy (XPS), respectively. Dynamic contact angle (DCA) was employed for wettability evaluation. One implant from each group was placed in the left tibia of twenty healthy, skeletally mature Santa Ines sheep (n=5). Bone area (BA) and bone-to-implant contact (BIC) were performed on thin sections (30μm) at 7, 14, 21 and 28days after implant installation. Despite the roughness and morphology similarities between the groups, at the XPS evaluation, the FA group presented 2.3 times less carbon on the surface (FN: 27.3% and FA: 11.6%), sharply enhanced hydrophilicity and significantly enhanced BA and BIC at 14, 21 and 28days of healing (P<0.05) compared with the FN. The data suggest that the hydrophilic FA accelerates the BA apposition and BIC interface around the implants during early stages of bone formation, providing highest degree of osseointegration.