Highly porous polycaprolactone microspheres for skeletal repair promote a mature bone cell phenotype in vitro.

Improving our ability to treat skeletal defects is a critical medical challenge that necessitates the development of new biomaterials. One promising approach involves the use of degradable polymer microparticles with an interconnected internal porosity. Here, we employed a double emulsion to generate such round microparticles (also known as microspheres) from a polycaprolactone-based polymerised high internal phase emulsion (polyHIPE). These microspheres effectively supported the growth of mesenchymal progenitors over a 30-day period, and when maintained in osteogenic media, cells deposited a bone-like extracellular matrix, as determined by histological staining for calcium and collagen. Interestingly, cells with an osteocyte-like morphology were observed within the core of the microspheres indicating the role of a physical environment comparable to native bone for this phenotype to occur. At later timepoints, these cultures had significantly increased mRNA expression of the osteocyte-specific markers dentin matrix phosphoprotein-1 (Dmp-1) and sclerostin, with sclerostin also observed at the protein level. Cells pre-cultured on porous microspheres exhibited enhanced survival rates compared to those pre-cultured on non-porous counterparts when injected. Cells precultured on both porous and non-porous microspheres promoted angiogenesis in a chorioallantoic membrane (CAM) assay. In summary, the polycaprolactone polyHIPE microspheres developed in this study exhibit significant promise as an alternative to traditional synthetic bone graft substitutes, offering a conducive environment for cell growth and differentiation, with the potential for better clinical outcomes in bone repair and regeneration.

Comparison of the Anabolic Effects of Reported Osteogenic Compounds on Human Mesenchymal Progenitor-derived Osteoblasts.

There is variability in the reported effects of compounds on osteoblasts arising from differences in experimental design and choice of cell type/origin. This makes it difficult to discern a compound's action outside its original study and compare efficacy between compounds. Here, we investigated five compounds frequently reported as anabolic for osteoblasts (17ß-estradiol (oestrogen), icariin, lactoferrin, lithium chloride, and menaquinone-4 (MK-4)) on human mesenchymal progenitors to assess their potential for bone tissue engineering with the aim of identifying a potential alternative to expensive recombinant growth factors such as bone morphogenetic protein 2 (BMP-2). Experiments were performed using the same culture conditions to allow direct comparison. The concentrations of compounds spanned two orders of magnitude to encompass the reported efficacious range and were applied continuously for 22 days. The effects on the proliferation (resazurin reduction and DNA quantification), osteogenic differentiation (alkaline phosphatase (ALP) activity), and mineralised matrix deposition (calcium and collagen quantification) were assessed. Of these compounds, only 10 µM MK-4 stimulated a significant anabolic response with 50% greater calcium deposition. Oestrogen and icariin had no significant effects, with the exception of 1 µM icariin, which increased the metabolic activity on days 8 and 22. 1000 µg/mL of lactoferrin and 10 mM lithium chloride both significantly reduced the mineralised matrix deposition in comparison to the vehicle control, despite the ALP activity being higher in lithium chloride-treated cells at day 15. This demonstrates that MK-4 is the most powerful stimulant of bone formation in hES-MPs of the compounds investigated, highlighting its potential in bone tissue engineering as a method of promoting bone formation, as well as its prospective use as an osteoporosis treatment.

Design and Evaluation of an Osteogenesis-on-a-Chip Microfluidic Device Incorporating 3D Cell Culture.

Microfluidic-based tissue-on-a-chip devices have generated significant research interest for biomedical applications, such as pharmaceutical development, as they can be used for small volume, high throughput studies on the effects of therapeutics on tissue-mimics. Tissue-on-a-chip devices are evolving from basic 2D cell cultures incorporated into microfluidic devices to complex 3D approaches, with modern designs aimed at recapitulating the dynamic and mechanical environment of the native tissue. Thus far, most tissue-on-a-chip research has concentrated on organs involved with drug uptake, metabolism and removal (e.g., lung, skin, liver, and kidney); however, models of the drug metabolite target organs will be essential to provide information on therapeutic efficacy. Here, we develop an osteogenesis-on-a-chip device that comprises a 3D environment and fluid shear stresses, both important features of bone. This inexpensive, easy-to-fabricate system based on a polymerized High Internal Phase Emulsion (polyHIPE) supports proliferation, differentiation and extracellular matrix production of human embryonic stem cell-derived mesenchymal progenitor cells (hES-MPs) over extended time periods (up to 21 days). Cells respond positively to both chemical and mechanical stimulation of osteogenesis, with an intermittent flow profile containing rest periods strongly promoting differentiation and matrix formation in comparison to static and continuous flow. Flow and shear stresses were modeled using computational fluid dynamics. Primary cilia were detectable on cells within the device channels demonstrating that this mechanosensory organelle is present in the complex 3D culture environment. In summary, this device aids the development of 'next-generation' tools for investigating novel therapeutics for bone in comparison with standard laboratory and animal testing.

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds.

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.

Composite porous scaffold of PEG/PLA support improved bone matrix deposition in vitro compared to PLA-only scaffolds.

Controllable pore size and architecture are essential properties for tissue-engineering scaffolds to support cell ingrowth colonization. To investigate the effect of polyethylene glycol (PEG) addition on porosity and bone-cell behavior, porous polylactic acid (PLA)-PEG scaffolds were developed with varied weight ratios of PLA-PEG (100/0, 90/10, 75/25) using solvent casting and porogen leaching. Sugar 200-300 µm in size was used as a porogen. To assess scaffold suitability for bone tissue engineering, MLO-A5 murine osteoblast cells were cultured and cell metabolic activity, alkaline phosphatase (ALP) activity and bone-matrix production determined using (alizarin red S staining for calcium and direct red 80 staining for collagen). It was found that metabolic activity was significantly higher over time on scaffolds containing PEG, ALP activity and mineralized matrix production were also significantly higher on scaffolds containing 25% PEG. Porous architecture and cell distribution and penetration into the scaffold were analyzed using SEM and confocal microscopy, revealing that inclusion of PEG increased pore interconnectivity and therefore cell ingrowth in comparison to pure PLA scaffolds. The results of this study confirmed that PLA-PEG porous scaffolds support mineralizing osteoblasts better than pure PLA scaffolds, indicating they have a high potential for use in bone tissue engineering applications. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1334-1340, 2018.