Time-lapsed imaging of nanocomposite scaffolds reveals increased bone formation in dynamic compression bioreactors.

Progress in bone scaffold development relies on cost-intensive and hardly scalable animal studies. In contrast to in vivo, in vitro studies are often conducted in the absence of dynamic compression. Here, we present an in vitro dynamic compression bioreactor approach to monitor bone formation in scaffolds under cyclic loading. A biopolymer was processed into mechanically competent bone scaffolds that incorporate a high-volume content of ultrasonically treated hydroxyapatite or a mixture with barium titanate nanoparticles. After seeding with human bone marrow stromal cells, time-lapsed imaging of scaffolds in bioreactors revealed increased bone formation in hydroxyapatite scaffolds under cyclic loading. This stimulatory effect was even more pronounced in scaffolds containing a mixture of barium titanate and hydroxyapatite and corroborated by immunohistological staining. Therefore, by combining mechanical loading and time-lapsed imaging, this in vitro bioreactor strategy may potentially accelerate development of engineered bone scaffolds and reduce the use of animals for experimentation.

Alginate dependent changes of physical properties in 3D bioprinted cell-laden porous scaffolds affect cell viability and cell morphology.

Three-dimensional (3D) cell-laden scaffolds are becoming more prevalent in bone tissue repair and regeneration. However, the influence of physical scaffold properties on cell behavior is still unclear. In this study, we fabricated four different alginate concentration (0.8, 1.3, 1.8 and 2.3%alg) composite cell-laden porous scaffolds using a 3D bioprinting technique. The aim was to investigate the changes of physical properties affected by the alginate concentration and the influences on cell behavior. The study showed that the different alginate concentration scaffolds had uniform macropores (500-600 µm) with compressive moduli ranging from 1.5 kPa (0.8%alg) to 14.2 kPa (2.3%alg). Long-term structural integrity of the printed scaffolds was achieved when cultured in cell culture media, but not when cultured in phosphate buffered saline (PBS). Scaffold structure, swelling behavior, and compressive moduli decreased with culturing time and higher alginate concentration lead to more stable physical scaffold properties. Meanwhile, human mesenchymal stem cell (hMSCs) laden non-printed and bioprinted composite scaffolds were fabricated. Bioprinting did not affect cell viability, but alginate concentration had a significant influence on cell viability and cell morphology. Lower alginate concentration scaffolds (0.8%alg) showed higher cell viability (84% ± 0.7% versus 68% ± 1.3%) compared to higher alginate concentration scaffolds (2.3%alg) at day 14. Live cell image in the 0.8%alg scaffolds demonstrated the formation of a 3D interconnected cellular network, while cells in the 1.8 and 2.3%alg scaffolds formed spheroids. In conclusion, this study broadens the design space for alginate-based bioinks for 3D bioprinting. Higher alginate concentration preserved better scaffold fidelity and mechanical properties. Better cell viability and cell spreading morphology was achieved in lower alginate concentration scaffolds, which is relevant for potential applications in bone tissue engineering.

Silk fibroin scaffolds with inverse opal structure for bone tissue engineering.

How scaffold porosity, pore diameter and geometry influence cellular behavior is-although heavily researched - merely understood, especially in 3D. This is mainly caused by a lack of suitable, reproducible scaffold fabrication methods, with processes such as gas foaming, lyophilization or particulate leaching still being the standard. Here we propose a method to generate highly porous silk fibroin scaffolds with monodisperse spherical pores, namely inverse opals, and study their effect on cell behavior. These silk fibroin inverse opal scaffolds were compared to salt-leached silk fibroin scaffolds in terms of human mesenchymal stem cell response upon osteogenic differentiation signals. While cell number remained similar on both scaffold types, extracellular matrix mineralization nearly doubled on the newly developed scaffolds, suggesting a positive effect on cell differentiation. By using the very same material with comparable average pore diameters, this increase in mineral content can be attributed to either the differences in pore diameter distribution or the pore geometry. Although the exact mechanisms leading to enhanced mineralization in inverse opals are not yet fully understood, our results indicate that control over pore geometry alone can have a major impact on the bioactivity of a scaffold toward stem cell differentiation into bone tissue. © 2016 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2074-2084, 2017.

The influence of curvature on three-dimensional mineralized matrix formation under static and perfused conditions: an in vitro bioreactor model.

Bone remodelling is the continuous turnover of bone by resorption and formation. It is controlled by interstitial fluid flow sensed by osteocytes. The refilling of bone resorption sites has been shown to be curvature driven. In vitro, curvature influences tissue growth and cytoskeletal arrangements under static and perfused conditions. Nevertheless, this has only been demonstrated for non-mineralized tissue in limited three-dimensional volumes. This study aims at investigating the influence of three different channel curvatures (S, -2.00 mm-1; M, -1.33 mm-1; L, -0.67 mm-1) on mineralized tissue formation in three dimensions under static and perfused conditions. The ingrowth of mineralized tissue into the channels was dependent on curvature and was higher under perfusion (M and S channels). L channels were not closed in any group compared with partially (static) or fully (perfused) closed M and S channels. Mineralized tissue morphology was cortical-like in static samples and trabecular-like in perfused samples. Our results suggest that the three-dimensional in vitro model presented is not only able to reveal effects of curvature on mineralized tissue formation, but may be used as an in vitro model for critical size defects in trabecular or cortical bone.

Effect of fetal bovine serum on mineralization in silk fibroin scaffolds.

Fetal bovine serum (FBS) is a common media supplement used in tissue engineering (TE) cultures. The chemical composition of FBS is known to be highly variable between different brands, types or batches and can have a significant impact on cell function. This study investigated the influence of four different FBS types in osteogenic or control medium on mineralization of acellular and cell-seeded silk fibroin (SF) scaffolds. In bone TE, mineralized tissue is considered as the final product of a successful cell culture. Calcium assays and micro-computed tomography scans revealed spontaneous mineralization on SF scaffolds with certain FBS types, even without cells present. In contrast, cell-mediated mineralization was found under osteogenic conditions only. Fourier transform infrared spectroscopy analysis demonstrated a similar ion composition of the mineralization present in scaffolds, whether cell-mediated or spontaneous. These results were confirmed by scanning electron microscopy. This study shows clear evidence for the influence of FBS type on mineralization on SF scaffolds. The suitability of FBS medium supplementation in TE studies is highly questionable with regard to reproducibility of studies and comparability of obtained results. For future TE studies, alternatives to conventional FBS such as defined FBS or serum-free media should be considered, as suggested decades ago.

Injectable and porous PLGA microspheres that form highly porous scaffolds at body temperature.

Injectable scaffolds are of interest in the field of regenerative medicine because of their minimally invasive mode of delivery. For tissue repair applications, it is essential that such scaffolds have the mechanical properties, porosity and pore diameter to support the formation of new tissue. In the current study, porous poly(dl-lactic acid-co-glycolic acid) (PLGA) microspheres were fabricated with an average size of 84±24µm for use as injectable cell carriers. Treatment with ethanolic sodium hydroxide for 2min was observed to increase surface porosity without causing the microsphere structure to disintegrate. This surface treatment also enabled the microspheres to fuse together at 37°C to form scaffold structures. The average compressive strength of the scaffolds after 24h at 37°C was 0.9±0.1MPa, and the average Young's modulus was 9.4±1.2MPa. Scaffold porosity levels were 81.6% on average, with a mean pore diameter of 54±38µm. This study demonstrates a method for fabricating porous PLGA microspheres that form solid porous scaffolds at body temperature, creating an injectable system capable of supporting NIH-3T3 cell attachment and proliferation in vitro.

Therapeutic potential of adipose-derived stromal cells in age-related osteoporosis.

Adipose-derived stromal cells (ASCs) are increasingly being used for orthopedic-based tissue engineering approaches due to their ability to readily undergo osteogenic differentiation. In the present study, we used in vitro and in vivo approaches to evaluate the use of ASCs as a treatment strategy for age-related osteoporosis. Molecular, histological and micro-computed tomography (micro-CT) based approaches confirmed that ASCs isolated from 18-week-old osteoporotic senescence-accelerated mice (SAMP6) were capable of undergoing osteogenesis when cultured in either silk fibroin (SF) scaffolds or scaffold-free microtissues (ASC-MT). A single intratibial injection of CM-Dil-labeled isogeneic ASCs or ASC-MT into SAMP6 recipients significantly improved trabecular bone quality after 6 weeks in comparison to untreated contralateral bones, as determined by micro-CT. Injected ASCs could be observed in paraffin wax bone sections at 24 h and 6 weeks post treatment and induced a significant increase in several molecular markers of bone turnover. Furthermore, a significant improvement in the osteogenic potential of osteoporotic patient-derived human bone marrow stromal cells (BMSCs) was observed when differentiated in conditioned culture media harvested from osteoporotic patient-derived human ASCs. These findings therefore support the use of ASCs as an autologous cell-based approach for the treatment of osteoporosis.

Pressureless mechanical induction of stem cell differentiation is dose and frequency dependent.

Movement is a key characteristic of higher organisms. During mammalian embryogenesis fetal movements have been found critical to normal tissue development. On the single cell level, however, our current understanding of stem cell differentiation concentrates on inducing factors through cytokine mediated biochemical signaling. In this study, human mesenchymal stem cells and chondrogenesis were investigated as representative examples. We show that pressureless, soft mechanical stimulation precipitated by the cyclic deformation of soft, magnetic hydrogel scaffolds with an external magnetic field, can induce chondrogenesis in mesenchymal stem cells without any additional chondrogenesis transcription factors (TGF-ß1 and dexamethasone). A systematic study on the role of movement frequency revealed a classical dose-response relationship for human mesenchymal stem cells differentiation towards cartilage using mere mechanical stimulation. This effect could even be synergistically amplified when exogenous chondrogenic factors and movement were combined.

Two-layer membranes of calcium phosphate/collagen/PLGA nanofibres: in vitro biomineralisation and osteogenic differentiation of human mesenchymal stem cells.

The present study evaluates the in vitro biomedical performance of an electrospun, flexible, anisotropic bilayer with one layer containing a collagen to mineral ratio similar to that in bone. The double membrane consists of a poly(lactide-co-glycolide) (PLGA) layer and an amorphous calcium phosphate (a-CaP)/collagen (Col)/PLGA layer. In vitro biomineralisation and a cell culture study with human mesenchymal stem cells (hMSC) were conducted to characterise such membranes for possible application as biomaterials. Nanofibres with different a-CaP/Col/PLGA compositions were synthesised by electrospinning to mimic the actual composition of bone tissue. Immersion in simulated body fluid and in cell culture medium resulted in the deposition of a hydroxyapatite layer. Incubation of hMSC for 4 weeks allowed for assessment of the proliferation and osteogenic differentiation of the cells on both sides of the double membrane. Confocal laser scanning microscopy was used to observe the proper adhesion of the cells. Calcium and collagen content was proven by Alizarin red S and Sirius red assays. Acute cytotoxic effects of the nanoparticles or the chemicals used in the scaffold preparation could be excluded based on viability assays (alamarBlue and alkaline phosphatase activity). The findings suggest possible application of such double membranes is in treatment of bone defects with complex geometries as wound dressing material.