Tissue Culture Plastic Controls Research Articles

Pre-clinical evaluation of advanced nerve guide conduits using a novel 3D in vitro testing model.

Autografts are the current gold standard for large peripheral nerve defects in clinics despite the frequently occurring side effects like donor site morbidity. Hollow nerve guidance conduits (NGC) are proposed alternatives to autografts, but failed to bridge gaps exceeding 3 cm in humans. Internal NGC guidance cues like microfibres are believed to enhance hollow NGCs by giving additional physical support for directed regeneration of Schwann cells and axons. In this study, we report a new 3D in vitro model that allows the evaluation of different intraluminal fibre scaffolds inside a complete NGC. The performance of electrospun polycaprolactone (PCL) microfibres inside 5 mm long polyethylene glycol (PEG) conduits were investigated in neuronal cell and dorsal root ganglion (DRG) cultures in vitro. Z-stack confocal microscopy revealed the aligned orientation of neuronal cells along the fibres throughout the whole NGC length and depth. The number of living cells in the centre of the scaffold was not significantly different to the tissue culture plastic (TCP) control. For ex vivo analysis, DRGs were placed on top of fibre-filled NGCs to simulate the proximal nerve stump. In 21 days of culture, Schwann cells and axons infiltrated the conduits along the microfibres with 2.2 ± 0.37 mm and 2.1 ± 0.33 mm, respectively. We conclude that this in vitro model can help define internal NGC scaffolds in the future by comparing different fibre materials, composites and dimensions in one setup prior to animal testing.

Laser microgrooving and resorbable blast texturing for enhanced surface function of titanium alloy for dental implant applications

Long-term dental implant success is dependent on biocompatibility and osseointegration between the bone and the implant. Surface modifications such as laser-induced microgrooving which increase contact area can enhance osseointegration by establishing and directing a stable attachment between the implant surface and peri‑implant bone. The objective of this study was to evaluate pre-osteoblast proliferation, morphology, and differentiation on titanium alloy (Ti64) surfaces—Laser-Lok© (LL), resorbable blast textured (RBT), and machined (M)—compared to tissue culture plastic (TCP) control. We hypothesized the LL surfaces would facilitate increased cellular alignment compared to all other groups, and LL and RBT surfaces would demonstrate enhanced proliferation and differentiation compared to M and TCP surfaces. Surface roughness was quantified using a surface profilometer, and water contact angle was measured to evaluate the hydrophilicity of the surfaces. Cellular function was assessed using quantitative viability and differentiation assays and image analyses, along with qualitative fluorescent (viability and cytoskeletal) imaging and scanning electron microscopy. No differences in surface roughness were observed between groups. Water contact angle indicated LL was the least hydrophilic surface, with RBT and M surfaces exhibiting greater hydrophilicity. Cell proliferation on day 2 was enhanced on both LL and RBT surfaces compared to M, and all three groups had higher cell numbers on day 2 compared to day 1. Cell orientation was driven by the geometry of the surface modification, as cells were more highly aligned on LL surfaces compared to TCP (on day 2) and RBT (on day 3). At day 21, cell proliferation was greater on LL, RBT, and TCP surfaces compared to M, though no differences in osteogenic differentiation were observed. Collectively, our results highlight the efficacy of laser microgrooved and resorbable blast textured surface modifications of Ti64 for enhancing cellular functions, which may facilitate improved osseointegration of dental implants.

A tunable physiomimetic stretch system evaluated with precision cut lung slices and recellularized human lung scaffolds.

Breathing exposes lung cells to continual mechanical stimuli, which is part of the microenvironmental signals directing cellular functions together with the extracellular matrix (ECM). Therefore, developing systems that incorporate both stimuli is urgent to fully understand cell behavior. This study aims to introduce a novel in vitro culture methodology combining a cyclic stretch that simulates in vivo breathing with 3D cell culture platforms in the form of decellularized lung slices (DLS) and precision cut lung slices (PCLS). To this end, we have constructed a device that mimics the amplitudes and frequencies of distensions seen in the breathing human lung. For its validation, we cultured H441 lung epithelial cells in human DLS exposed to 16 stretch cycles per minute with a 10% stretch amplitude. Cell viability (resazurin reduction), proliferation (Ki-67) and YAP1 activation were evaluated at 24 and 96 h by immunohistochemistry, while the expression of SFTPB, COL3A1, COL4A3 and LAMA5 was evaluated by qPCR. Cyclic stretch induced an increase in SFTPB expression after 24 h without a concomitant increase in the stretch responsive gene YAP1. Moreover, the ECM milieu lowered the expression of the basement membrane protein genes COL4A3 and LAMA5 compared to tissue culture plastic control cultures, but no effect was observed by the mechanical stimuli. The device also confirmed good compatibility with PCLS culture, showing preserved morphology and metabolism in rat PCLS after 72 h of mechanical stretch. Thus, we present a novel device and methodology for the easy assembling and study of lung tissue slice cultures subjected to physiomimetic mechanical stimuli, which shows promise for future studies of cell and tissue function in a lung ECM milieu with physiological or pathological mechanical stimuli.

TiO2 Nanostructures That Reduce the Infectivity of Human Respiratory Viruses Including SARS-CoV-2.

The rapid emergence and global spread of the COVID-19 causing Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and its subsequent mutated strains has caused unprecedented health, economic, and social devastation. Respiratory viruses such as SARS-CoV-2 can be transmitted through both direct and indirect channels, including aerosol respiratory droplets, contamination of inanimate surfaces (fomites), and direct person-to-person contact. Current methods of virus inactivation on surfaces include chemicals and biocides, and while effective, continuous and repetitive cleaning of all surfaces is not always viable. Recent work in the field of biomaterials engineering has established the antibacterial effects of hydrothermally synthesized TiO2 nanostructured surfaces against both Gram-negative and -positive bacteria. The current study investigates the effectiveness of said TiO2 nanostructured surfaces against two enveloped human coronaviruses, SARS-CoV-2 and HCoV-NL63, and nonenveloped HRV-16 for surface-based inactivation. Results show that structured surfaces reduced infectious viral loads of SARS-CoV-2 (5 log), HCoV-NL63 (3 log), and HRV-16 (4 log) after 5 h, compared to nonstructured and tissue culture plastic control surfaces. Interestingly, infectious virus remained present on control tissue culture plastic after 7 h exposure. These encouraging results establish the potential use of nanostructured surfaces to reduce the transmission and spread of both enveloped and nonenveloped respiratory viruses, by reducing their infectious period on a surface. The dual antiviral and antibacterial properties of these surfaces support their potential application in a wide variety of settings such as hospitals and healthcare environments, public transport and community hubs.

Three-dimensional Printing of Biomimetic Titanium Mimicking Trabecular Bone Induces Human Mesenchymal Stem Cell Proliferation: An In-vitro Analysis.

In vitro analysis. The aim of this study was to assess the effect of three-dimensional (3D) printing of porous titanium on human mesenchymal stem cell (hMSC) adhesion, proliferation, and osteogenic differentiation. A proprietary implant using three-dimensional porous titanium (3D-pTi) that mimics trabecu-lar bone structure, roughness, porosity, and modulus of elasticity was created (Ti-LIFE technology™, Spineart SA Switzerland). Such implants may possess osteoinductive properties augmenting fusion in addition to their structural advantages. However, the ability of 3D-pTi to affect in vitro cellular proliferation and osteogenic differentiation remains undefined. Disks of 3D-pTi with a porosity of 70% to 75% and pore size of 0.9 mm were produced using additive manufacturing technology. 2D Ti6Al4V (2D-Ti) and 2D polyetheretherketone (2D-PEEK) disks were prepared using standard manufacturing process. Tissue culture plastic (TCP) served as the control surface. All discs were characterized using 2D-micros-copy, scanning electron microscopy (SEM), and x-ray micro-computed tomography. Forty thousand hMSCs were seeded on the disks and TCP and cultured for 42 days. hMSC morphology was assessed using environmental SEM and confocal imaging following phalloidin staining. hMSC proliferation was evaluated using DNA fluorescent assay. hMSC differentiation was assessed using RT-qPCR for genes involved in hMSC osteogenic differentiation and biochemical assays were performed for alkaline phosphatase activity (ALP) and calcium content. 3D-pTi lead to a higher cell number as compared to 2D-Ti and 2D-PEEK at D21, D28 and D42. ALP activity of hMSCs seeded into 3D-pTi scaffolds was as high as or higher than that of hMSCs seeded onto TCP controls over all time points and consistently higher than that of hMSCs seeded onto 2D-Ti scaffolds. However, when ALP activity was normalized to protein content, no statistical differences were found between all scaffolds tested and TCP controls. 3D-pTi provides a scaffold for bone formation that structurally mimics cancellous bone and improves hMSC adhesion and proliferation compared to 2D-Ti and PEEK.

Dermal Extracellular Matrix-Derived Hydrogels as an In Vitro Substrate to Study Mast Cell Maturation.

Mast cells (MCs) are pro-inflammatory tissue-resident immune cells that play a key role in inflammation. MCs circulate in peripheral blood as progenitors and undergo terminal differentiation in the tissue microenvironment where they can remain for many years. This in situ maturation results in tissue- and species-specific MC phenotypes, culminating in significant variability in response to environmental stimuli. There are many challenges associated with studying mature tissue-derived MCs, particularly in humans. In cases where cultured MCs are able to differentiate in two-dimensional in vitro cultures, there remains an inability for full maturation. Extracellular matrix (ECM) scaffolds provide for a more physiologically relevant environment for cells in vitro and have been shown to modulate the response of other immune cells such as T cells, monocytes, and macrophages. To improve current in vitro testing platforms of MCs and to assess future use of ECM scaffolds for MC regulation, we studied the in vitro response of human MCs cultured on decellularized porcine dermis hydrogels (dermis extracellular matrix hydrogel [dECM-H]). This study investigated the effect of dECM-H on cellular metabolic activity, cell viability, and receptor expression compared to collagen type I hydrogel (Collagen-H). Human MCs showed different metabolic activity when cultured in the dECM-H and also upregulated immunoglobulin E (IgE) receptors associated with MC maturation/activation compared to collagen type I. These results suggest an overall benefit in the long-term culture of human MCs in the dECM-H compared to Collagen-H providing important steps toward a model that is more representative of in vivo conditions. Graphical abstract [Formula: see text] Impact statement Mast cells (MCs) are difficult to culture in vitro as current culture conditions and substrates fail to promote similar phenotypic features observed in vivo. Extracellular matrix (ECM)-based biomaterials offer three-dimensional, tissue-specific environments that more closely resemble in vivo conditions. Our study explores the use of dermal ECM hydrogels for MC culture and shows significant upregulation of metabolic activity, cell viability, and gene expression of markers associated with MC maturation or activation compared to collagen type I-hydrogel and tissue culture plastic controls at 7 days. These results are among the first to describe MC behavior in response to ECM hydrogels.

Si-Fe-C-N Coatings for Biomedical Applications: A Combinatorial Approach.

Ceramic coatings may prolong the lifetime of joint implants. Certain ions and wear debris may however lead to negative biological effects. SiN-based materials may substantially reduce these effects, but still need optimization for the application. In this study, a combinatorial deposition method enabled an efficient evaluation of a range of Si–Fe–C–N coating compositions on the same sample. The results revealed compositional gradients of Si (26.0–33.9 at.%), Fe (9.6–20.9 at.%), C (8.2–13.9 at.%) and N (39.7–47.2 at.%), and low oxygen contaminations (0.3–0.6 at.%). The mechanical properties varied with a hardness (H) ranging between 13.7–17.3 GPa and an indentation modulus (M) between 190–212 GPa. Both H and M correlated with the Si (H and M increased as Si increased) and Fe (H and M decreased as Fe increased) content. A slightly columnar morphology was observed in cross-sections, as well as a surface roughness in the nm range. A cell study revealed adhering pre-osteogenic MC3T3 cells, with a morphology similar to that of cells seeded on a tissue culture plastic control. The investigated coatings could be considered for further investigation due to the ability to tune their mechanical properties while maintaining a smooth surface, together with their promising in vitro cell response.

Stressful Surfaces: Cell Metabolism on a Poorly Adhesive Substrate.

The adhesion and proliferation of cells are exquisitely sensitive to the nature of the surface to which they attach. Aside from cell counting, cell "health" on surfaces is typically established by measuring the metabolic rate with dyes that participate in the metabolic pathway or using "live/dead" assays with combinations of membrane permeable/impermeable dyes. The binary information gleaned from these tests-whether cells are attached or not, and whether they are living or dead-provides an incomplete picture of cell health. In the present work, proliferation rates and net metabolism of 3T3 fibroblasts seeded on "biocompatible" ultrathin polyelectrolyte multilayer films and on control tissue culture plastic were compared. Cells adhered to, and proliferated on, both surfaces, which were shown to be nontoxic according to live/dead assays. However, adhesion was poorer on the multilayer surface, illustrated by diffuse organization of the actin cytoskeleton and less-developed focal adhesions. Proliferation was also slower on the multilayer. When normalized for the total number of cells, it was shown that cells on multilayers experienced a five-day burst of metabolic stress, after which the metabolic rate approached that of the control surface. This initial state of high stress has not been reported or appreciated in studies of cell growth on multilayers, although the observation period for this system is usually a few days.

Comparison of Two Porcine Collagen Membranes Combined with rhBMP-2 and rhBMP-9 on Osteoblast Behavior In Vitro.

To investigate the bone-inducing properties of two types of collagen membranes in combination with recombinant human bone morphogenetic protein (rhBMP)-2 and rhBMP-9 on osteoblast behavior. Porcine pericardium collagen membranes (PPCM) and porcine dermis-derived collagen membranes (PDCM) were coated with either rhBMP-2 or rhBMP-9. The adsorption and release abilities were first investigated via enzyme-linked immunosorbent assay up to 10 days. Moreover, murine bone stromal ST2 cell adhesion, proliferation, and osteoblast differentiation were assessed by MTS assay; real-time polymerase chain reaction for genes encoding runt-related transcription factor 2 (Runx2); alkaline phosphatase (ALP); and osteocalcin, ALP assay, and alizarin red staining. Both rhBMP-2 and rhBMP-9 adsorbed to collagen membranes and were gradually released over time up to 10 days. PPCM showed significantly less cell attachment, whereas PDCM demonstrated comparable cell attachment with the control tissue culture plastic at 8 hours. While both rhBMPs were shown not to affect cell proliferation, collagen membranes combined with rhBMP-9 significantly increased ALP activity at 7 days and ALP mRNA levels at either 3 or 14 days compared with the control tissue culture plastic. Furthermore, rhBMP-9 increased osteocalcin mRNA levels and alizarin red staining at 14 days compared with the control tissue culture plastic. The results from this study suggest that both porcine-derived collagen membranes combined with rhBMP-9 accelerated the osteopromotive potential of ST2 cells. Interestingly, rhBMP-9 demonstrated additional osteogenic differentiation compared with rhBMP-2 and may serve as a suitable growth factor for future clinical use.

Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or β-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.

Effects of an injectable platelet-rich fibrin on osteoblast behavior and bone tissue formation in comparison to platelet-rich plasma

Platelet-rich plasma (PRP) has been utilized for many years as a regenerative agent capable of inducing vascularization of various tissues using blood-derived growth factors. Despite this, drawbacks mostly related to the additional use of anti-coagulants found in PRP have been shown to inhibit the wound healing process. For these reasons, a novel platelet concentrate has recently been developed with no additives by utilizing lower centrifugation speeds. The purpose of this study was therefore to investigate osteoblast behavior of this novel therapy (injectable-platelet-rich fibrin; i-PRF, 100% natural with no additives) when compared to traditional PRP. Human primary osteoblasts were cultured with either i-PRF or PRP and compared to control tissue culture plastic. A live/dead assay, migration assay as well as a cell adhesion/proliferation assay were investigated. Furthermore, osteoblast differentiation was assessed by alkaline phosphatase (ALP), alizarin red and osteocalcin staining, as well as real-time PCR for genes encoding Runx2, ALP, collagen1 and osteocalcin. The results showed that all cells had high survival rates throughout the entire study period irrespective of culture-conditions. While PRP induced a significant 2-fold increase in osteoblast migration, i-PRF demonstrated a 3-fold increase in migration when compared to control tissue-culture plastic and PRP. While no differences were observed for cell attachment, i-PRF induced a significantly higher proliferation rate at three and five days when compared to PRP. Furthermore, i-PRF induced significantly greater ALP staining at 7 days and alizarin red staining at 14 days. A significant increase in mRNA levels of ALP, Runx2 and osteocalcin, as well as immunofluorescent staining of osteocalcin was also observed in the i-PRF group when compared to PRP. In conclusion, the results from the present study favored the use of the naturally-formulated i-PRF when compared to traditional PRP with anti-coagulants. Further investigation into the direct role of fibrin and leukocytes contained within i-PRF are therefore warranted to better elucidate their positive role in i-PRF on tissue wound healing.

In vitro effects of hyaluronic acid on human periodontal ligament cells

BackgroundHyaluronic acid (HA) has been reported to have a positive effect on periodontal wound healing following nonsurgical and surgical therapy. However, to date, a few basic in vitro studies have been reported to investigating the potential of HA on human periodontal ligament (PDL) cell regeneration. Therefore, the aim of this study was to investigate the effect of HA on PDL cell compatibility, proliferation, and differentiation in vitro.MethodsEither non-cross-linked (HA_ncl) or cross-linked (HA_cl) HA was investigated. Human PDL cells were seeded in 7 conditions as follows (1) Control tissue culture plastic (TCP) (2) dilution of HA_ncl (1:100), (3) dilution of HA_ncl (1:10), 4) HA_ncl directly coated onto TCP, (5) dilution of HA_cl (1:100), 6) dilution of HA_cl (1:10) and (7) HA_cl directly coated onto TCP. Samples were then investigated for cell viability using a live/dead assay, an inflammatory reaction using real-time PCR and ELISA for MMP2, IL-1 and cell proliferation via an MTS assay. Furthermore, the osteogenic potential of PDL cells was assessed by alkaline phosphatase(ALP) activity, collagen1(COL1) and osteocalcin(OCN) immunostaining, alizarin red staining, and real-time PCR for genes encoding Runx2, COL1, ALP, and OCN.ResultsBoth HA_ncl and HA_cl showed high PDL cell viability (greater than 90%) irrespective of the culturing conditions. Furthermore, no significant difference in both mRNA and protein levels of proinflammatory cytokines, including MMP2 and IL-1 expression was observed. Both diluted HA_ncl and HA_cl significantly increased cell numbers compared to the controlled TCP samples at 3 and 5 days. HA_ncl and HA_cl in standard cell growth media significantly decreased ALP staining, COL1 immunostaining and down-regulated early osteogenic differentiation, including Runx2, COL1, and OCN mRNA levels when compared to control samples. When osteogenic differentiation medium (ODM) was added, interestingly, the expression of early osteogenic markers increased by demonstrating higher levels of COL1 and ALP expression; especially in HA 1:10 diluted condition. Late stage osteogenic markers remained inhibited.ConclusionsBoth non-cross-linked and cross-linked HA maintained high PDL cell viability, increased proliferation, and early osteogenic differentiation. However, HA was consistently associated with a significant decrease in late osteogenic differentiation of primary human PDL cells. Future in vitro and animal research is necessary to further characterize the effect of HA on periodontal regeneration.

Electrohydrodynamic printing as a method to micropattern large titanium implant surfaces with photocrosslinkable structures

Metallic implants are widely used in orthopaedic and orthodontic applications. However, generally surface treatment of the metallic surfaces is necessary to render them more biologically active. Herein, we describe a direct write printing method to modify metallic implant surfaces with biocompatible polymers with microscale precision. Application of polymeric micropatterns on metallic implant surfaces can (i) improve their interaction with the host tissue, (ii) enable the delivery of growth factors, antibiotics, anti-inflammatory cytokines etc from the implant surface and (iii) can control the immune responses to the implant via controlling the attachment of immune cells, such as macrophages. Surface patterns with a resolution of less than 50 μm can be created using an electro hydrodynamic (EHD) printing, a template-free and single-step process. We present a revised EHD printing method for the deposition of parallel strips of photocrosslinkable, cell adhesive polymeric composites with spacing of around 20 μm onto medical grade titanium substrates. Optimization of voltage, feeding rate and collection speed resulted in regular structures via very rapid movement of the grounded rotating collector driven to equivalent of the linear surface speed of above 100 cm s−1. In the experimental part a mixture of chemically modified PEG /gelatin was deposited onto a conductive titanium substrate with different surface pretreatments with an area of 400 mm2. Acid etched or UV treated titanium surfaces improved the stability of the printed structures. Polymeric lines induced temporary orientation of human monocytes (THP-1) and induced a thicker cell multilayer formation by 3T3 fibroblasts (p < 0.05). Staining of the monocytes for M1(CD80) and M2 (CD206) macrophage markers on the patterned surface showed mixed populations with higher anti-inflammatory cytokine secretion compared to tissue culture plastic control. The results demonstrate the suitability of this method for the preparation of biomaterials with structured surfaces on large areas and with desired chemical composition.

Hyaluronic Acid Gel-Based Scaffolds as Potential Carrier for Growth Factors: An In Vitro Bioassay on Its Osteogenic Potential.

Hyaluronic acid (HA) has been utilized for a variety of regenerative medical procedures due to its widespread presence in connective tissue and perceived biocompatibility. The aim of the present study was to investigate HA in combination with recombinant human bone morphogenetic protein 9 (rhBMP9), one of the most osteogenic growth factors of the BMP family. HA was first combined with rhBMP9 and assessed for the adsorption and release of rhBMP9 over 10 days by ELISA. Thereafter, ST2 pre-osteoblasts were investigated by comparing (1) control tissue culture plastic, (2) HA alone, and (3) HA with rhBMP9 (100 ng/mL). Cellular proliferation was investigated by a MTS assay at one, three and five days and osteoblast differentiation was investigated by alkaline phosphatase (ALP) activity at seven days, alizarin red staining at 14 days and real-time PCR for osteoblast differentiation markers. The results demonstrated that rhBMP9 adsorbed within HA scaffolds and was released over a 10-day period in a controlled manner. While HA and rhBMP9 had little effect on cell proliferation, a marked and pronounced effect was observed for cell differentiation. rhBMP9 significantly induced ALP activity, mRNA levels of collagen1α2, and ALP and osteocalcin (OCN) at three or 14 days. HA also demonstrated some ability to induce osteoblast differentiation by increasing mRNA levels of OCN and increasing alizarin red staining at 14 days. In conclusion, the results from the present study demonstrate that (1) HA may serve as a potential carrier for various growth factors, and (2) rhBMP9 is a potent and promising inducer of osteoblast differentiation. Future animal studies are now necessary to investigate this combination approach in vivo.

Structural, thermal, in vitro degradation and cytocompatibility properties of P2O5-B2O3-CaO-MgO-Na2O-Fe2O3 glasses

Borophosphate glasses with compositions of (48−x)P2O5-(12+x)B2O3-14CaO-20MgO-1Na2O-5Fe2O3 (where x=0, 3, 8mol%) were prepared via a melt-quenching process. The effects of replacing P2O5 with B2O3 on the structural, thermal, degradation properties and cytocompatibility were investigated. Fourier transform infrared (FTIR) spectroscopy analysis confirmed the existence of BO3 triangular units and BO4 tetrahedral units within all the glasses with an increase of B/P ratio from 0.25 to 0.5. The BO4 units within the glass structure were observed to cause an increase in density (ρ) as well as glass transition (Tg) temperature and to decrease the crystallisation temperature (Tc). A decrease in thermal stability which indicated by process window was also observed in the case of substitution of P2O5 with B2O3. Degradation analysis of the glasses indicated that the dissolution rate increased with the addition of B2O3. The decrease in the thermal stability and chemical durability were attributed to the increase of BO3 units, which could increase crystallisation tendency and be easily hydrolysed by solution. The effect of boron addition on the cytocompatibility of the glasses was analysed using Alamar Blue and alkaline phosphatase (ALP) assays and DNA quantification. MG63 osteosarcoma cells cultured in direct contact with the glass samples surface for 14days showed better cytocompatibility, compared to the tissue culture plastic (TCP) control group. In summary, the glass formulation with 12mol% B2O3 presented the best cytocompatibility and thermal stability, thus could be considered for continuous fibre fabrication in future research and downstream activities.

In vitro characterization of an osteoinductive biphasic calcium phosphate in combination with recombinant BMP2

BackgroundThe repair of alveolar bone defects with growth factors and bone grafting materials has played a pivotal role in modern dentistry. Recombinant human bone morphogenetic protein-2 (rhBMP2), an osteoinductive growth factor capable of cell recruitment and differentiation towards the osteoblast lineage, has been utilized in combination with various biomaterials to further enhance new bone formation. Recently, a group of novel biphasic calcium phosphate (BCP) bone grafting materials have been demonstrated to possess osteoinductive properties by demonstrating signs of ectopic bone formation. The aim of the present study was to study the effects of rhBMP2 in combination with osteoinductive BCP bone grafts on osteoblast cell behaviour.MethodsMC3T3-E1 pre-osteoblasts were seeded on 1) control tissue culture plastic, 2) 10 mg of BCP alone, 3) 100 ng rhBMP2, and 4) 100 ng rhBMP2+ 10 mg of BCP and analyzed for cell recruitment via a Transwell chamber, proliferation via an MTS assay and differentiation as assessed by alkaline phosphatase (ALP) activity, alizarin red staining and real-time PCR for osteoblast differentiation markers including Runx2, collagen1, ALP, and osteocalcin (OCN).ResultsrhBMP2 was able to significantly upregulate cell recruitment whereas the addition of BCP as well as BCP alone had no additional ability to improve osteoblast recruitment. Both BCP and rhBMP2 were able to significantly increase cell proliferation at 3 and 5 days post seeding and cell number was further enhanced when rhBMP2 was combined with BCP. In addition, the combination of rhBMP2 with BCP significantly improved ALP activity at 7 and 14 days post seeding, alizarin red staining at 14 days, and mRNA levels of Runx2, ALP and osteocalcin when compared to cells seeded with rhBMP2 alone or BCP alone.ConclusionsThe results from the present study demonstrate that 1) the osteoinductive potential of BCP bone particles is equally as osteopromotive as rhBMP2 on in vitro osteoblast differentiation and 2) BCP particles in combination with rhBMP2 is able to further increase the osteopromotive differentiation of osteoblasts in vitro when compared to either rhBMP2 alone or BCP alone. Future animal testing is further required to investigate this combination approach on new bone formation.

A TiO2 - polyurethane composite as an alternative ventricular catheter material

Event Abstract Back to Event A TiO2 - polyurethane composite as an alternative ventricular catheter material Davide Erbogasto1, Dimitrios Lamprou2 and Richard A. Black1 1 University of Strathclyde, Biomedical Engineering, United Kingdom 2 University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Science, United Kingdom Introduction: Ventricular shunt catheters for the treatment of hydrocephalus are commonly made from poly (di-methyl) siloxane (PDMS), the walls of which are perforated with holes for cerebro-spinal fluid (CSF) to pass through. Although considerable improvements in design have been made since their introduction all CSF shunts in use today have a high incidence of failure, with infection and shunt obstruction being the most serious. Titanium dioxide is renowned for its photocatalytic properties, which gives rise to greater hydrophilicity and production of free radicals upon exposure to UV light. Such properties have been widely employed to confer antifouling and bacteriostatic properties to other materials, and are particularly appealing for this application; indeed, the use of a micro-porous catheter with increased hydrophilicity has been proposed as a means to decrease the failure rate[1],[2]. Materials and Methods: Titanium (IV) dioxide was synthesized in situ via a sol-gel reaction starting from an alkoxide precursor (Ti(OnBu)4) mixed with acetic acid, which was then mixed with a poly(caprolactone) solution and electrospun onto the surface of an electrospun polyurethane substrate (Z6A1, Biomer Technology Ltd., UK). Similar titanium dioxide-loaded polyurethane samples were prepared. Three weight ratios polymer:titania precursor were chosen (1/7, 1/5, 1/3). The thermal properties and surface morphology of both cast and electrospun forms have been characterized via Differential Scanning Calorimetry (DSC) and Atomic Force Microscopy (AFM). The viability of mammalian cells (5H-SY5Y, human neuroblastoma cell line, PHE, UK), seeded at 12.5·103 cells/well was measured after 2 and 4 days via an absorbance assay (alamarBlue®, ABDserotec, UK). Cell viability was expressed as the percentage reduction of the dye relative to PDMS and tissue culture plastic controls, respectively. Results and Discussion: Addition of titanium dioxide alter the physical-chemical properties of the polymeric matrix, as shown in Fig. 1: the melting point shifts towards lower temperatures and the peak broadens with increasing TiO2 content. TiO2 seems to be well dispersed throughout the polymeric matrix and in an amorphous phase. Upon UV irradiation, titanium dioxide triggers photocatalytic reactions which increase the hydrophilic character of the surface and generate free radicals[3]. Dynamic cultures of 5H-SY5Y cells show an effective reduction in cell viability over the time scale considered on the hybrid materials, while no significant change is noted on the pure electrospun materials (Fig. 2). Conclusion: Titanium dioxide-hybrid materials have a clear effect on reducing viability of cells seeded on them. Further studies are required to establish the precise contribution of individual factors, such as fibre morphology, mechanical properties, enhanced wettability and photocatalytic activity. The authors wish to acknowledge the support of the UK Engineering & Physical Sciences Research Council (EPSRC) Doctoral Training Centre in Medical Devices, University of Strathclyde (EPSRC Grant Ref. EP/F50036X/1) for the studentship awarded to DE.

Preparation, characterisation, and in vitro evaluation of electrically conducting poly(ɛ-caprolactone)-based nanocomposite scaffolds using PC12 cells.

In the current study, we describe the synthesis, material characteristics, and cytocompatibility of conducting poly (ɛ-caprolactone) (PCL)-based nano-composite films. Electrically conducting carbon nano-fillers (carbon nano-fiber (CNF), nano-graphite (NG), and liquid exfoliated graphite (G)) were used to prepare porous film type scaffolds using modified solvent casting methods. The electrical conductivity of the nano-composite films was increased when carbon nano-fillers were incorporated in the PCL matrix. CNF-based nano-composite films showed the highest increase in electrical conductivity. The presence of an ionic solution significantly improved the conductivity of some of the polymers, however at least 24 h was required to absorb the simulated ion solutions. CNF-based nano-composite films were found to have good thermo-mechanical properties compared to other conducting polymer films due to better dispersion and alignment in the critical direction. Increased nano-filler content increased the crystallisation temperature. Analysis of cell viability revealed no increase in cell death on any of the polymers compared to tissue culture plastic controls, or compared to PCL polymer without nano-composites. The scaffolds showed some variation when tested for PC12 cell attachment and proliferation, however all the polymers supported PC12 attachment and differentiation in the absence of cell adhesion molecules. In general, CNF-based nano-composite films with highest electrical conductivity and moderate roughness showed highest cell attachment and proliferation. These polymers are promising candidates for use in neural applications in the area of bionics and tissue engineering due to their unique properties.

Thermoresponsive Substrates Used for the Growth and Controlled Differentiation of Human Mesenchymal Stem Cells.

This communication outlines the advances made in the development of thermoresponsive substrates for human mesenchymal stem cell (hMSC) expansion and subsequent controlled specific and multilineage differentiation from a previous study performed by this group. Previously, the development of an inexpensive and technically accessible method for hMSC expansion and harvesting was reported, using the solvent casting deposition method and thermoresponsive poly(N-isopropylacrylamide). Here, the logical continuation of this work is reported with the multipassage expansion of hMSCs with phenotypic maintenance followed by induced adipogenic, osteogenic, and chondrogenic differentiation. Interestingly, 1 μm thick solvent cast films are not only capable of hosting an expanding population of phenotypically preserved hMSCs similar to tissue culture plastic controls, but also the cells detached via temperature control better maintain their ability to differentiate compared to conventionally trypsinized cells. This approach to hMSC expansion and differentiation can be highly attractive to stem cell researchers where clinical therapies have seen a collective deviation away from the employment of animal derived products such as proteolytic trypsin.

Tubular Scaffold with Shape Recovery Effect for Cell Guide Applications.

Tubular scaffolds with aligned polylactic acid (PLA) fibres were fabricated for cell guide applications by immersing rolled PLA fibre mats into a polyvinyl acetate (PVAc) solution to bind the mats. The PVAc solution was also mixed with up to 30 wt % β-tricalcium phosphate (β-TCP) content. Cross-sectional images of the scaffold materials obtained via scanning electron microscopy (SEM) revealed the aligned fibre morphology along with a significant number of voids in between the bundles of fibres. The addition of β-TCP into the scaffolds played an important role in increasing the void content from 17.1% to 25.3% for the 30 wt % β-TCP loading, which was measured via micro-CT (µCT) analysis. Furthermore, µCT analyses revealed the distribution of aggregated β-TCP particles in between the various PLA fibre layers of the scaffold. The compressive modulus properties of the scaffolds increased from 66 MPa to 83 MPa and the compressive strength properties decreased from 67 MPa to 41 MPa for the 30 wt % β-TCP content scaffold. The scaffolds produced were observed to change into a soft and flexible form which demonstrated shape recovery properties after immersion in phosphate buffered saline (PBS) media at 37 °C for 24 h. The cytocompatibility studies (using MG-63 human osteosarcoma cell line) revealed preferential cell proliferation along the longitudinal direction of the fibres as compared to the control tissue culture plastic. The manufacturing process highlighted above reveals a simple process for inducing controlled cell alignment and varying porosity features within tubular scaffolds for potential tissue engineering applications.