3D Bioprinting of Human Tissues: Biofabrication, Bioinks, and Bioreactors.

The field of tissue engineering has progressed tremendously over the past few decades in its ability to fabricate functional tissue substitutes for regenerative medicine and pharmaceutical research. Conventional scaffold-based approaches are limited in their capacity to produce constructs with the functionality and complexity of native tissue. Three-dimensional (3D) bioprinting offers exciting prospects for scaffolds fabrication, as it allows precise placement of cells, biochemical factors, and biomaterials in a layer-by-layer process. Compared with traditional scaffold fabrication approaches, 3D bioprinting is better to mimic the complex microstructures of biological tissues and accurately control the distribution of cells. Here, we describe recent technological advances in bio-fabrication focusing on 3D bioprinting processes for tissue engineering from data processing to bioprinting, mainly inkjet, laser, and extrusion-based technique. We then review the associated bioink formulation for 3D bioprinting of human tissues, including biomaterials, cells, and growth factors selection. The key bioink properties for successful bioprinting of human tissue were summarized. After bioprinting, the cells are generally devoid of any exposure to fluid mechanical cues, such as fluid shear stress, tension, and compression, which are crucial for tissue development and function in health and disease. The bioreactor can serve as a simulator to aid in the development of engineering human tissues from in vitro maturation of 3D cell-laden scaffolds. We then describe some of the most common bioreactors found in the engineering of several functional tissues, such as bone, cartilage, and cardiovascular applications. In the end, we conclude with a brief insight into present limitations and future developments on the application of 3D bioprinting and bioreactor systems for engineering human tissue.

Mechanoregulation analysis of bone formation in tissue engineered constructs requires a volumetric method using time-lapsed micro-computed tomography.

Bone can adapt its microstructure to mechanical loads through mechanoregulation of the (re)modeling process. This process has been investigated in vivo using time-lapsed micro-computed tomography (micro-CT) and micro-finite element (FE) analysis using surface-based methods, which are highly influenced by surface curvature. Consequently, when trying to investigate mechanoregulation in tissue engineered bone constructs, their concave surfaces make the detection of mechanoregulation impossible when using surface-based methods. In this study, we aimed at developing and applying a volumetric method to non-invasively quantify mechanoregulation of bone formation in tissue engineered bone constructs using micro-CT images and FE analysis. We first investigated hydroxyapatite scaffolds seeded with human mesenchymal stem cells that were incubated over 8 weeks with one mechanically loaded and one control group. Higher mechanoregulation of bone formation was measured in loaded samples with an area under the curve for the receiver operating curve (AUCformation) of 0.633-0.637 compared to non-loaded controls (AUCformation: 0.592-0.604) during culture in osteogenic medium (p < 0.05). Furthermore, we applied the method to an in vivo mouse study investigating the effect of loading frequencies on bone adaptation. The volumetric method detected differences in mechanoregulation of bone formation between loading conditions (p < 0.05). Mechanoregulation in bone formation was more pronounced (AUCformation: 0.609-0.642) compared to the surface-based method (AUCformation: 0.565-0.569, p < 0.05). Our results show that mechanoregulation of formation in bone tissue engineered constructs takes place and its extent can be quantified with a volumetric mechanoregulation method using time-lapsed micro-CT and FE analysis. STATEMENT OF SIGNIFICANCE: Many efforts have been directed towards optimizing bone scaffolds for tissue growth. However, the impact of the scaffolds mechanical environment on bone growth is still poorly understood, requiring accurate assessment of its mechanoregulation. Existing surface-based methods were unable to detect mechanoregulation in tissue engineered constructs, due to predominantly concave surfaces in scaffolds. We present a volumetric approach to enable the precise and non-invasive quantification and analysis of mechanoregulation in bone tissue engineered constructs by leveraging time-lapsed micro-CT imaging, image registration, and finite element analysis. The implications of this research extend to diverse experimental setups, encompassing culture conditions, and material optimization, and investigations into bone diseases, enabling a significant stride towards comprehensive advancements in bone tissue engineering and regenerative medicine.

Physiological cell bioprinting density in human bone-derived cell-laden scaffolds enhances matrix mineralization rate and stiffness under dynamic loading.

Human organotypic bone models are an emerging technology that replicate bone physiology and mechanobiology for comprehensive in vitro experimentation over prolonged periods of time. Recently, we introduced a mineralized bone model based on 3D bioprinted cell-laden alginate-gelatin-graphene oxide hydrogels cultured under dynamic loading using commercially available human mesenchymal stem cells. In the present study, we created cell-laden scaffolds from primary human osteoblasts isolated from surgical waste material and investigated the effects of a previously reported optimal cell printing density (5 × 106 cells/mL bioink) vs. a higher physiological cell density (10 × 106 cells/mL bioink). We studied mineral formation, scaffold stiffness, and cell morphology over a 10-week period to determine culture conditions for primary human bone cells in this microenvironment. For analysis, the human bone-derived cell-laden scaffolds underwent multiscale assessment at specific timepoints. High cell viability was observed in both groups after bioprinting (>90%) and after 2 weeks of daily mechanical loading (>85%). Bioprinting at a higher cell density resulted in faster mineral formation rates, higher mineral densities and remarkably a 10-fold increase in stiffness compared to a modest 2-fold increase in the lower printing density group. In addition, physiological cell bioprinting densities positively impacted cell spreading and formation of dendritic interconnections. We conclude that our methodology of processing patient-specific human bone cells, subsequent biofabrication and dynamic culturing reliably affords mineralized cell-laden scaffolds. In the future, in vitro systems based on patient-derived cells could be applied to study the individual phenotype of bone disorders such as osteogenesis imperfecta and aid clinical decision making.

Coating of metal implant materials with strontium.

The aim of this study was to show that cathodic polarization can be used for coating commercial implant surfaces with an immobilized but functional and bioavailable surface layer of strontium (Sr). Moreover, this study assessed the effect of fluorine on Sr-attachment. X-ray photoelectron spectroscopy revealed that addition of fluorine (F) to the buffer during coating increased surface Sr-amounts but also changed the chemical surface composition by adding SrF2 alongside of SrO whereas pre-treatment of the surface by pickling in hydrofluoric acid appeared to hinder Sr-attachment. Assessment of the bio-availability hinted at a positive effect of Sr on cell differentiation given that the surface reactivity of the original surface remained unchanged. Additional SrF2 on the surface appeared to reduce undesired surface contamination while maintaining the surface micro-topography and micro-morphology. Anyhow, this surface modification revealed to create nano-nodules on the surface.

Decreased bone mineral density and reduced bone quality in H(+) /K(+) ATPase beta-subunit deficient mice.

Proton pump inhibitors (PPIs) are widely used against gastroesophageal reflux disease. Recent epidemiological studies suggest that PPI users have an increased risk of fractures, but a causal relationship has been questioned. We have therefore investigated the skeletal phenotype in H(+) /K(+) ATPase beta-subunit knockout (KO) female mice. Skeletal parameters were determined in 6- and 20-month-old KO mice and in wild-type controls (WT). Whole body bone mineral density (BMD) and bone mineral content (BMC) were measured by dual energy X-ray absorptiometry (DXA). Femurs were examined with µCT analyses and break force were examined by a three-point bending test. Plasma levels of gastrin, RANKL, OPG, osteocalcin, leptin, and PTH were analyzed. KO mice had lower whole body BMC at 6 months (0.53 vs. 0.59 g, P = 0.035) and at 20 months (0.49 vs. 0.74 g, P < 0.01) compared to WT as well as lower BMD at 6 months (0.068 vs. 0.072 g/cm(2) , P = 0.026) and 20 months (0.067 vs. 0.077 g/cm(2) , P < 0.01). Mechanical strength was lower in KO mice at the age of 20 months (6.7 vs. 17.9 N, P < 0.01). Cortical thickness at 20 months and trabecular bone volume% at 6 months were significantly reduced in KO mice. Plasma OPG/RANKL ratio and PTH was increased in KO mice compared to controls. H(+) /K(+) ATPase beta subunit KO mice had decreased BMC and BMD, reduced cortical thickness and inferior mechanical bone strength. Whereas the mechanism is uncertain, these findings suggest a causal relationship between long-term PPI use and an increased risk of fractures.

Multi-doped apatite: Strontium, magnesium, gallium and zinc ions synergistically affect osteogenic stimulation in human mesenchymal cells important for bone tissue engineering.

Functional calcium phosphate biomaterials can be designed as carriers of a balanced mixture of biologically relevant ions able to target critical processes in bone regeneration. They hold the potential to use mechanisms very similar to growth factors naturally produced during fracture healing, while circumventing some of their drawbacks. Here we present a novel phase of carbonated-apatite containing Mg2+, Sr2+, Zn2+ and Ga3+ ions (HApMgSrZnGa). While all dopants decrease the crystallinity, Ga3+ limits crystal growth and enables the formation of a nanosized apatite phase with enhanced specific surface area. Coexistence of the ions enhances degradability and controls solubility of low crystalline, distorted, multi-doped apatite structure, controlled by Ga3+ ions accumulated at the surface. Consequently, HApMgSrZnGa supports the viability of human mesenchymal stromal cells (MSCs) and induces their stimulation along the osteogenic lineage. In addition, the co-released ions has a synergistic antimicrobial effect, particularly within the HApMgSrZnGa-Au(arg) composite with Au(arg) as contact-based antimicrobial. The activity is stable up to two months in vitro. Osteogenic nature and antimicrobial activity, combined in a single biomaterial, are suggesting a well-balanced, multi-doped apatite design applicable as future option in bone regeneration and tissue engineering.

Long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids.

Mechanical loading has been shown to influence various osteogenic responses of bone-derived cells and bone formationin vivo. However, the influence of mechanical stimulation on the formation of bone organoidin vitrois not clearly understood. Here, three-dimensional (3D) bioprinted human mesenchymal stem cells-laden graphene oxide composite scaffolds were cultured in a novel cyclic-loading bioreactors for up to 56 d. Our results showed that mechanical loading from day 1 (ML01) significantly increased organoid mineral density, organoid stiffness, and osteoblast differentiation compared with non-loading and mechanical loading from day 21. Importantly, ML01 stimulated collagen I maturation, osteocyte differentiation, lacunar-canalicular network formation and YAP expression on day 56. These finding are the first to reveal that long-term mechanical loading is required for the formation of 3D bioprinted functional osteocyte bone organoids. Such 3D bone organoids may serve as a human-specific alternative to animal testing for the study of bone pathophysiology and drug screening.

Bioactive Interpenetrating Hydrogel Networks Based on 2-Hydroxyethyl Methacrylate and Gelatin Intertwined with Alginate and Dopped with Apatite as Scaffolding Biomaterials.

Our goal was to create bioimitated scaffolding materials for biomedical purposes. The guiding idea was that we used an interpenetrating structural hierarchy of natural extracellular matrix as a "pattern" to design hydrogel scaffolds that show favorable properties for tissue regeneration. Polymeric hydrogel scaffolds are made in a simple, environmentally friendly way without additional functionalization. Gelatin and 2-hydroxyethyl methacrylate were selected to prepare interpenetrating polymeric networks and linear alginate chains were added as an interpenetrant to study their influence on the scaffold's functionalities. Cryogelation and porogenation methods were used to obtain the designed scaffolding biomaterials. The scaffold's structural, morphological, and mechanical properties, in vitro degradation, and cell viability properties were assessed to study the effects of the preparation method and alginate loading. Apatite as an inorganic agent was incorporated into cryogelated scaffolds to perform an extensive biological assay. Cryogelated scaffolds possess superior functionalities essential for tissue regeneration: fully hydrophilicity, degradability and mechanical features (2.08-9.75 MPa), and an optimal LDH activity. Furthermore, cryogelated scaffolds loaded with apatite showed good cell adhesion capacity, biocompatibility, and non-toxic behavior. All scaffolds performed equally in terms of metabolic activity and osteoconductivity. Cryogelated scaffolds with/without HAp could represent a new advance to promote osteoconductivity and enhance hard tissue repair. The obtained series of scaffolding biomaterials described here can provide a wide range of potential applications in the area of biomedical engineering.

3D bioprinting of graphene oxide-incorporated cell-laden bone mimicking scaffolds for promoting scaffold fidelity, osteogenic differentiation and mineralization.

Bioprinting is a promising technique for facilitating the fabrication of engineered bone tissues for patient-specific defect repair and for developing in vitro tissue/organ models for ex vivo tests. However, polymer-based ink materials often result in insufficient mechanical strength, low scaffold fidelity and loss of osteogenesis induction because of the intrinsic swelling/shrinking and bioinert properties of most polymeric hydrogels. Here, we developed a human mesenchymal stem cells (hMSCs)-laden graphene oxide (GO)/alginate/gelatin composite bioink to form 3D bone-mimicking scaffolds using a 3D bioprinting technique. Our results showed that the GO composite bioinks (0.5GO, 1GO, 2GO) with higher GO concentrations (0.5, 1 and 2 mg/ml) improved the bioprintability, scaffold fidelity, compressive modulus and cell viability at day 1. The higher GO concentration increased the cell body size and DNA content, but the 2GO group swelled and had the lowest compressive modulus at day 42. The 1GO group had the highest osteogenic differentiation of hMSC with the upregulation of osteogenic-related gene (ALPL, BGLAP, PHEX) expression. To mimic critical-sized calvarial bone defects in mice and prove scaffold fidelity, 3D cell-laden GO defect scaffolds with complex geometries were successfully bioprinted. 1GO maintained the best scaffold fidelity and had the highest mineral volume after culturing in the bioreactor for 42 days. In conclusion, GO composite bioinks had better bioprintability, scaffold fidelity, cell proliferation, osteogenic differentiation and ECM mineralization than the pure alginate/gelatin system. The optimal GO group was 1GO, which demonstrated the potential for 3D bioprinting of bone tissue models and tissue engineering applications.

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.

Biodegradable Hydrogel Scaffolds Based on 2-Hydroxyethyl Methacrylate, Gelatin, Poly(ß-amino esters), and Hydroxyapatite.

New composite 3D scaffolds were developed as a combination of synthetic polymer, poly(2-hydroxyethyl methacrylate) (PHEMA), and a natural polymer, gelatin, with a ceramic component, nanohydroxyapatite (ID nHAp) dopped with metal ions. The combination of a synthetic polymer, to be able to tune the structure and the physicochemical and mechanical properties, and a natural polymer, to ensure the specific biological functions of the scaffold, with inorganic filler was applied. The goal was to make a new material with superior properties for applications in the biomedical field which mimics as closely as possible the native bone extracellular matrix (ECM). Biodegradable PHEMA hydrogel was obtained by crosslinking HEMA by poly(ß-amino esters) (PBAE). The scaffold's physicochemical and mechanical properties, in vitro degradation, and biological activity were assessed so to study the effects of the incorporation of nHAp in the (PHEMA/PBAE/gelatin) hydrogel, as well as the effect of the different pore-forming methods. Cryogels had higher elasticity, swelling, porosity, and percent of mass loss during degradation than the samples obtained by porogenation. The composite scaffolds had a higher mechanical strength, 10.14 MPa for the porogenated samples and 5.87 MPa for the cryogels, but a slightly lower degree of swelling, percent of mass loss, and porosity than the hybrid ones. All the scaffolds were nontoxic and had a high cell adhesion rate, which was 15-20% higher in the composite samples. Cell metabolic activity after 2 and 7 days of culture was higher in the composites, although not statistically different. After 28 days, cell metabolic activity was similar in all scaffolds and the TCP control. No effect of integrating nHAp into the scaffolds on osteogenic cell differentiation could be observed. Synergetic effects occurred which influenced the mechanical behavior, structure, physicochemical properties, and interactions with biological species.

Scaffold Pore Geometry Guides Gene Regulation and Bone-like Tissue Formation in Dynamic Cultures.

Cells sense and respond to scaffold pore geometry and mechanical stimuli. Many fabrication methods used in bone tissue engineering render structures with poorly controlled pore geometries. Given that cell-scaffold interactions are complex, drawing a conclusion on how cells sense and respond to uncontrolled scaffold features under mechanical loading is difficult. In this study, monodisperse templated scaffolds (MTSC) were fabricated and used as well-defined porous scaffolds to study the effect of dynamic culture conditions on bone-like tissue formation. Human bone marrow-derived stromal cells were cultured on MTSC or conventional salt-leached scaffolds (SLSC) for up to 7 weeks, either under static or dynamic conditions (wall shear stress [WSS] using spinner flask bioreactors). The influence of controlled spherical pore geometry of MTSC subjected to static or dynamic conditions on osteoblast cell differentiation, bone-like tissue formation, structure, and distribution was investigated. WSS generated within the two idealized geometrical scaffold features was assessed. Distinct response to fluid flow in osteoblast cell differentiation were shown to be dependent on scaffold pore geometry. As revealed by collagen staining and microcomputed tomography images, dynamic conditions promoted a more regular extracellular matrix (ECM) formation and mineral distribution in both scaffold types compared with static conditions. The results showed that regulation of bone-related genes and the amount and the structure of mineralized ECM were dependent on scaffold pore geometry and the mechanical cues provided by the two different culture conditions. Under dynamic conditions, SLSC favored osteoblast cell differentiation and ECM formation, whereas MTSC enhanced ECM mineralization. The spherical pore shape in MTSC supported a more trabecular bone-like structure under dynamic conditions compared with MTSC statically cultured or to SLSC under either static or dynamic conditions. These results suggest that cell activity and bone-like tissue formation is driven not only by the pore geometry but also by the mechanical environment. This should be taken into account in the future design of complex scaffolds, which should favor cell differentiation while guiding the formation, structure, and distribution of the engineered bone tissue. This could help to mimic the anatomical complexity of the bone tissue structure and to adapt to each bone defect needs. Impact statement Aging of the human population leads to an increasing need for medical implants with high success rate. We provide evidence that cell activity and the amount and structure of bone-like tissue formation is dependent on the scaffold pore geometry and on the mechanical environment. Fabrication of complex scaffolds comprising concave and planar pore geometries might represent a promising direction toward the tunability and mimicry the structural complexity of the bone tissue. Moreover, the use of fabrication methods that allow a systematic fabrication of reproducible and geometrically controlled structures would simplify scaffold design optimization.

Rosuvastatin promotes osteoblast differentiation and regulates SLCO1A1 transporter gene expression in MC3T3-E1 cells.

Rosuvastatin (RSV) is a synthetic statin with favourable pharmacologic properties including minimal metabolism, hepatic selectivity and enhanced inhibition of HMG-CoA reductase. An induction of osteoblast differentiation has been reported in vitro with lipophilic statins but not with RSV, which, like pravastatin, is relatively hydrophilic compared with other statins. To mediate its action, an active transport mechanism via solute carrier (SLC) transporters from the SLC16, SLC21/SLCO and SLC22 gene family - specifically Slc16a1, Slco1a1, Slco2b1 and Slc22a8 - may be present to allow effective entry in osteoblastic cells. In this study, we demonstrate that RSV induced osteoblast differentiation, as measured by increased BMP-2 gene expression and secretion, and ALP activity in MC3T3-E1 osteoblast cells, without significantly affecting cell proliferation within the concentration range of 0.001-10 µM. Low concentrations of RSV (0.001-0.01 µM) were protective against cell death whereas higher concentrations (10-100 µM) showed cytotoxicity. Moreover, MC3T3-E1 osteoblasts expressed high levels of Slco1a1 and Slc16a1 mRNA and low levels of Slco2b1 and Slc22a8 mRNA, when compared with kidney and liver tissues from mice. Slco1a1 gene expression increased 12-fold during osteoblast differentiation and was further regulated after RSV treatment. In conclusion, as for other statins, RSV promotes osteoblast differentiation, and also, demonstrated for the first time, regulates the expression of Slco1a1, which may constitute the transport system for RSV across the cell membrane in mature osteoblasts.

Optimization of mechanical stiffness and cell density of 3D bioprinted cell-laden scaffolds improves extracellular matrix mineralization and cellular organization for bone tissue engineering.

Bioprinting is an emerging technology in which cell-laden biomaterials are precisely dispersed to engineer artificial tissues that mimic aspects of the anatomical and structural complexity of relatively soft tissues such as skin, vessels, and cartilage. However, reproducing the highly mineralized and cellular diversity of bone tissue is still not easily achievable and is yet to be demonstrated. Here, an extrusion-based 3D bioprinting strategy is utilized to fabricate 3D bone-like tissue constructs containing osteogenic cellular organization. A simple and low-cost bioink for 3D bioprinting of bone-like tissue is prepared based on two unmodified polymers (alginate and gelatin) and combined with human mesenchymal stem cells (hMSCs). To form 3D bone-like tissue and bone cell phenotype, the influence of different scaffold stiffness and cell density of 3D bioprinted cell-laden porous scaffolds on osteogenic differentiation and bone-like tissue formation was investigated over time. Our results showed that soft scaffolds (0.8%alg, 0.66 ± 0.08 kPa) had higher DNA content, enhanced ALP activity and stimulated osteogenic differentiation than stiff scaffolds (1.8%alg, 5.4 ± 1.2 kPa). At day 42, significantly more mineralized tissue was formed in soft scaffolds than in stiff scaffolds (43.5 ± 7.1 mm3 vs. 22.6 ± 6.0 mm3). Importantly, immunohistochemistry staining demonstrated more osteocalcin protein expression in high mineral compared to low mineral regions. Additionally, cells in soft scaffolds exhibited osteoblast- and early osteocyte-related gene expression and 3D cellular network within the mineralized matrix at day 42. Furthermore, the results showed that cell density in 15 M cells/ml can promote cell-cell connections at day 7 and mineral formation at day 14, while 5 M cells/ml had the significantly higher mineral formation rate than 15 M cells/ml from day 14 to day 21. In summary, this work reports the formation of 3D bioprinted bone-like tissue using a simple and low-cost cell-laden bioink, which was optimized for stiffness and cell density, showing great promise for bone tissue engineering applications. STATEMENT OF SIGNIFICANCE: In this study, we presented for the first time a framework combining 3D bioprinting, bioreactor system and time-lapsed micro-CT monitoring to provide in vitro scaffold fabrication, maturation, and mineral visualization for bone tissue engineering. 3D bone-like tissue constructs have been formed via optimizing scaffold stiffness and cell density. The soft scaffolds had higher cell proliferation, enhanced alkaline phosphatase activity and stimulated osteogenic differentiation with 3D cellular network foramtion than stiff scaffolds. Significantly more mineralized bone-like tissue was formed in soft scaffolds than stiff scaffolds at day 42. Meanwhile, cell density in 15 M cells/ml can promote cell-cell connections and mineral formation in 14 days, while the higher mineral formation rate was found in 5 M cells/ml from day 14 to day 21.

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.

hiPS-MSCs differentiation towards fibroblasts on a 3D ECM mimicking scaffold.

Fibroblasts are ubiquitous cells that constitute the stroma of virtually all tissues and play vital roles in homeostasis. The poor innate healing capacity of fibroblastic tissues is attributed to the scarcity of fibroblasts as collagen-producing cells. In this study, we have developed a functional ECM mimicking scaffold that is capable to supply spatial allocation of stem cells as well as anchorage and storage of growth factors (GFs) to direct stem cells differentiate towards fibroblasts. Electrospun PCL fibers were embedded in a PEG-fibrinogen (PF) hydrogel, which was infiltrated with connective tissue growth factor (CTGF) to form the 3D nanocomposite PFP-C. The human induced pluripotent stem cells derived mesenchymal stem cells (hiPS-MSCs) with an advance in growth over adult MSCs were applied to validate the fibrogenic capacity of the 3D nanocomposite scaffold. The PFP-C scaffold was found not only biocompatible with the hiPS-MSCs, but also presented intriguingly strong fibroblastic commitments, to an extent comparable to the positive control, tissue culture plastic surfaces (TCP) timely refreshed with 100% CTGF. The novel scaffold presented not only biomimetic ECM nanostructures for homing stem cells, but also sufficient cell-approachable bio-signaling cues, which may synergistically facilitate the control of stem cell fates for regenerative therapies.

Simvastatin-activated implant surface promotes osteoblast differentiation in vitro.

The bone growth promoting effects of statins suggest that these bioactive molecules can be used to improve the integration of bone-anchored implants. This study aimed at the application of simvastatin with dental implants for use in patients with low bone density. Coin-shaped titanium zirconium samples with grit-blasted and acid-etched surface were coated with simvastatin, using a novel anodic oxidation setup under alkaline conditions. The presence of intact simvastatin attached to the surface was confirmed by infrared spectroscopy. A binding site on the aliphatic O-H group was discovered and the integration of (1)H, (18)O and (12)C in the depth of the surface were observed by secondary ion mass spectroscopy. A simvastatin concentration of about 60 g/cm(2) was found in a release study over 72 h. The simvastatin-coated surfaces promoted alkaline phosphatase, collagen type I and osteocalcin gene expression of MC3T3-E1 cells. This suggested that the demonstrated coating holds potential for use in patients with compromised bone.

Electrospun PCL/PEO coaxial fibers for basic fibroblast growth factor delivery.

The poor innate healing capacity of fibroblastic tissues, such as pelvic floor fascia, is attributed to the scarcity of fibroblasts to produce collagen, as the main collagen producing cells. Coaxial electrospun PCL/PEO fibers containing basic fibroblast growth factor (FGF-2) were evaluated for the local and temporal delivery of FGF-2 for promoting fibroblast proliferation. PCL/PEO coaxial fibers with a highly porous surface were successfully developed using coaxial electrospinning. The diameter of the PCL/PEO microfibers produced by coaxial electrospinning could be tuned by the electrospinning parameters. XPS surface chemistry probing and CA wettability analysis confirmed that the outer surface of the coaxial fibers is PCL. The protein was successfully encapsulated and a sustained release was observed over more than 9 days. In vitro, PCL/PEO coaxial fibers supported fibroblast cell adhesion. In addition, PCL/PEO coaxial fibers containing FGF-2 significantly enhanced fibroblast cell viability and proliferation. Further, Coll-I expression was significantly expressed after day 1 while down-regulated after day 9 compared to the control group. These results indicate that coaxial polymeric fibers allow local and sustained growth factor delivery with prolonged efficacy and longevity for connective tissue regeneration.

Ultraporous interweaving electrospun microfibers from PCL-PEO binary blends and their inflammatory responses.

Production of one dimensional nanomaterials with secondary morphology exhibiting unique functions is challenging. Here we report for the first time that a nanoscale immiscible polymer blend solution electrojet can assemble into ultraporous interweaving microfibers. This intriguingly novel morphology originated from a blend of polycaprolactone (PCL) and polyethylene oxide (PEO) in a DCM-DMF mixed solution when the ratio between each component reached a threshold and when the electrospinning parameters were delicately controlled. The morphology, crystallinity, surface chemistry and wettabilities were characterized to understand the mechanism of formation. The interplay of the two semi-crystalline polymers and the pair of solvents/non-solvents with the electrospinning processing parameters was found to be critical for the formation of the unique structure. Furthermore, the interesting combination of biocompatible, biodegradable PCL with protein-resistant PEO motivated us to assess its inflammation responses on the RAW 264.7 macrophage cell line. All fibers were found to be biocompatible with low inflammation potential upon incubation, while compared with pure PCL nanofibers; the unique interweaving microfibers induced a slightly higher inflammatory reaction.