Bone Tissue Engineering Approaches Research Articles

Improving pore size of electrospun gelatin scaffolds containing graphene oxide using PEG as a sacrificial agent for bone tissue engineering

In this study, polyethylene glycol (PEG), as a sacrificial agent, and gelatin containing graphene oxide (GO) are co-electrospun to prepare scaffolds. Based on pore size measurements, removal of PEG leads to an increase in the pore size of modified scaffolds, resulting in easier infiltration. Moreover, noticeable elastic modulus of GO increased the mechanical strength. Alizarin red staining indicated proliferation and differentiation of human bone marrow-derived mesenchymal stem cells seeded on the modified scaffolds. Thus, gelatin scaffold containing 0.5% GO with 20% PEG at the ratio of 80:20 for gelatin–PEG was selected as the optimal scaffold for bone tissue engineering approaches.

From brain to bone: Harnessing extracellular vesicles released from TBI to enhance osteogenesis by 3D-Printed hydrogel scaffold

The clinical indications reveal that traumatic brain injury (TBI) accelerates the process of limb bone regeneration. However, the mechanisms underlying this phenomenon remain ambiguous. In this inquiry, the activation of endogenous neural stem cells in the hippocampus post TBI led to the secretion of extracellular vesicles (NSC-EV) that targeted bone marrow mesenchymal stem cells, ultimately promoting bone healing. The enrichment of miR-9-5p in NSC-EV facilitated osteogenesis through the activation of the MAP3K3 pathway. We also present a novel hydrogel formulation for clinical application of NSC-EVs by means of ROS-responsive EV delivery. The hydrogel was cross-linked with EVs to create a water-based gel encapsulation, which was applied to a 3D-printed β-TCP scaffold for controlled long-term release that corresponded to those of physiological bone tissue. Our investigation represents the inaugural usage of EVs derived from neural stem cells in bone tissue engineering. These EVs were found to encourage information exchange between the central nervous system and bone tissue regeneration. Our composite scaffold demonstrated impressive bone regeneration qualities in rats with cranial defects, highlighting its potential for pioneering approaches in bone tissue engineering and innovative medical technologies for clinical applications.

A tissue engineered 3D printed calcium alkali phosphate bioceramic bone graft enables vascularization and regeneration of critical-size discontinuity bony defects in vivo.

Introduction: Recently, efforts towards the development of patient-specific 3D printed scaffolds for bone tissue engineering from bioactive ceramics have continuously intensified. For reconstruction of segmental defects after subtotal mandibulectomy a suitable tissue engineered bioceramic bone graft needs to be endowed with homogenously distributed osteoblasts in order to mimic the advantageous features of vascularized autologous fibula grafts, which represent the standard of care, contain osteogenic cells and are transplanted with the respective blood vessel. Consequently, inducing vascularization early on is pivotal for bone tissue engineering. The current study explored an advanced bone tissue engineering approach combining an advanced 3D printing technique for bioactive resorbable ceramic scaffolds with a perfusion cell culture technique for pre-colonization with mesenchymal stem cells, and with an intrinsic angiogenesis technique for regenerating critical size, segmental discontinuity defects in vivo applying a rat model. To this end, the effect of differing Si-CAOP (silica containing calcium alkali orthophosphate) scaffold microarchitecture arising from 3D powder bed printing (RP) or the Schwarzwalder Somers (SSM) replica fabrication technique on vascularization and bone regeneration was analyzed in vivo. In 80 rats 6-mm segmental discontinuity defects were created in the left femur. Methods: Embryonic mesenchymal stem cells were cultured on RP and SSM scaffolds for 7d under perfusion to create Si-CAOP grafts with terminally differentiated osteoblasts and mineralizing bone matrix. These scaffolds were implanted into the segmental defects in combination with an arteriovenous bundle (AVB). Native scaffolds without cells or AVB served as controls. After 3 and 6months, femurs were processed for angio-µCT or hard tissue histology, histomorphometric and immunohistochemical analysis of angiogenic and osteogenic marker expression. Results: At 3 and 6months, defects reconstructed with RP scaffolds, cells and AVB displayed a statistically significant higher bone area fraction, blood vessel volume%, blood vessel surface/volume, blood vessel thickness, density and linear density than defects treated with the other scaffold configurations. Discussion: Taken together, this study demonstrated that the AVB technique is well suited for inducing adequate vascularization of the tissue engineered scaffold graft in segmental defects after 3 and 6months, and that our tissue engineering approach employing 3D powder bed printed scaffolds facilitated segmental defect repair.

Assembling the Puzzle Pieces. Insights for in Vitro Bone Remodeling.

As a highly dynamic organ, bone changes during throughout a person's life. This process is referred to as 'bone remodeling' and it involves two stages - a well-balanced osteoclastic bone resorption and an osteoblastic bone formation. Under normal physiological conditions bone remodeling is highly regulated that ensures tight coupling between bone formation and resorption, and its disruption results in a bone metabolic disorder, most commonly osteoporosis. Though osteoporosis is one of the most prevalent skeletal ailments that affect women and men aged over 40 of all races and ethnicities, currently there are few, if any safe and effective therapeutic interventions available. Developing state-of-the-art cellular systems for bone remodeling and osteoporosis can provide important insights into the cellular and molecular mechanisms involved in skeletal homeostasis and advise better therapies for patients. This review describes osteoblastogenesis and osteoclastogenesis as two vital processes for producing mature, active bone cells in the context of interactions between cells and the bone matrix. In addition, it considers current approaches in bone tissue engineering, pointing out cell sources, core factors and matrices used in scientific practice for modeling bone diseases and testing drugs. Finally, it focuses on the challenges that bone regenerative medicine is currently facing.

Polyvinylpyrrolidone Nanofibers Incorporating Mesoporous Bioactive Glass for Bone Tissue Engineering.

Composite biomaterials that combine osteoconductive and osteoinductive properties are a promising approach for bone tissue engineering (BTE) since they stimulate osteogenesis while mimicking extracellular matrix (ECM) morphology. In this context, the aim of the present research was to produce polyvinylpyrrolidone (PVP) nanofibers containing mesoporous bioactive glass (MBG) 80S15 nanoparticles. These composite materials were produced by the electrospinning technique. Design of experiments (DOE) was used to estimate the optimal electrospinning parameters to reduce average fiber diameter. The polymeric matrices were thermally crosslinked under different conditions, and the fibers' morphology was studied using scanning electron microscopy (SEM). Evaluation of the mechanical properties of nanofibrous mats revealed a dependence on thermal crosslinking parameters and on the presence of MBG 80S15 particles inside the polymeric fibers. Degradation tests indicated that the presence of MBG led to a faster degradation of nanofibrous mats and to a higher swelling capacity. The assessment of in vitro bioactivity in simulated body fluid (SBF) was performed using MBG pellets and PVP/MBG (1:1) composites to assess if the bioactive properties of MBG 80S15 were kept when it was incorporated into PVP nanofibers. FTIR and XRD analysis along with SEM-EDS results indicated that a hydroxy-carbonate apatite (HCA) layer formed on the surface of MBG pellets and nanofibrous webs after soaking in SBF over different time periods. In general, the materials revealed no cytotoxic effects on the Saos-2 cell line. The overall results for the materials produced show the potential of the composites to be used in BTE.

Physiologic Response Evaluation of Human Foetal Osteoblast Cells within Engineered 3D-Printed Polylactic Acid Scaffolds.

Large bone defect treatments have always been one of the important challenges in clinical practice and created a huge demand for more efficacious regenerative approaches. The bone tissue engineering (BTE) approach offered a new alternative to conventional bone grafts, addressing all clinical needs. Over the past years, BTE research is focused on the study and realisation of new biomaterials, including 3D-printed supports to improve mechanical, structural and biological properties. Among these, polylactic acid (PLA) scaffolds have been considered the most promising biomaterials due to their good biocompatibility, non-toxic biodegradability and bioresorbability. In this work, we evaluated the physiological response of human foetal osteoblast cells (hFOB), in terms of cell proliferation and osteogenic differentiation, within oxygen plasma treated 3D-printed PLA scaffolds, obtained by fused deposition modelling (FDM). A mechanical simulation to predict their behaviour to traction, flexural or torque solicitations was performed. We found that: 1. hFOB cells adhere and grow on scaffold surfaces; 2. hFOB grown on oxygen plasma treated PLA scaffolds (PLA_PT) show an improvement of cell adhesion and proliferation, compared to not-plasma treated scaffolds (PLA_NT); 3. Over time, hFOB penetrate along strands, differentiate, and form a fibrous matrix, tissue-like; 4. 3D-printed PLA scaffolds have good mechanical behaviour in each analysed configuration. These findings suggest that 3D-printed PLA scaffolds could represent promising biomaterials for medical implantable devices in the orthopaedic field.

Photo-click crosslinked hydrogel containing MgO2-loaded PLGA microsphere with concurrent magnesium and oxygen release for bone tissue engineering

Overcoming hypoxia and providing a proper microenvironment for osteogenic differentiation are the most critical challenges in bone tissue engineering (BTE). Hydrogels with the capability to release oxygen and magnesium ions are considered to be a promising approach for BTE. Here, MgO2-loaded polylactic-co-glycolic acid (PLGA) microspheres were prepared to improve oxygen release, magnesium release, and pH control. Then, PLGA/MgO2 microspheres-loaded thiolated alginate (AlgT)/gelatin methacryloyl (GelMA) photocrosslinkable hydrogel was prepared using click reaction, and the effect of different concentrations of AlgT on the physical and chemical properties of the hydrogel was investigated. The pore size decreased as AlgT concentration increased from 101.11 ± 26.04 to 74.48 ± 14.03 μm, and the swelling ratio, elastic modulus, and compressive strength were increased when AlgT concentration increased from 821.30 ± 30% to 950 ± 60%, 0.69–1.23 MPa and 1.293–2.81 MPa, respectively. Pore size, swelling ratio, elastic modulus, and compressive strength were 88.28 ± 20 μm, 522.82 ± 29%, 1.64 ± 0.04 MPa, and 4.044 ± 0.041 MPa for microsphere-loaded hydrogel, respectively. Besides, the level of released magnesium and oxygen during 14 days ranged from 13 to 22 mg/L and 7.7–8.2 mg/L, respectively. The MTT and live/dead assay results showed suitable cell viability and adhesion on the microsphere-loaded hydrogels. Also, the calcium content and alkaline phosphatase activity of human Adipose-derived mesenchymal stem cells (hAMSCs) cultured on samples were investigated. Moreover, hAMSCs indicated osteogenic differentiation after 21 days in culture, including the expression of significant alkaline phosphatase, osteocalcin, and osteopontin. This study's results indicate the prepared hydrogels' suitability for osteogenic differentiation and bone tissue engineering.

Hyaluronic acid hydrogels support to generate integrated bone formation through endochondral ossification in vivo using mesenchymal stem cells.

Engineered cartilage tissue from differentiated mesenchymal stem cells (MSCs) can generate bone in vivo through endochondral ossification (ECO). This ECO-mediated approach has the potential to circumvent the severe problems associated with conventional MSC-based bone tissue engineering techniques that lack mechanisms to induce angiogenesis. Hyaluronic acid (HA) is a key component in the cartilage extracellular matrix. However, the ECO-supporting properties of HA remain largely unclear. This study aimed to compare the ability of HA and collagen hydrogels to support in vitro differentiation of MSC-based hypertrophic cartilage tissues and to promote endochondral bone formation in vivo. Following the chondrogenic and hypertrophic differentiation in vitro, both HA and collagen constructs accumulated sulfated glycosaminoglycan (sGAG) and type 1, type II, and type X collagen. However, HA hydrogels exhibited a more uniform distribution of sGAG, type 1 collagen, type X collagen, and osteocalcin proteins; in addition, the cells embedded in the hydrogels had more rounded cell morphologies than those in the collagen constructs. At week 5 of in vitro culture, two to three constructs were implanted into a subcutaneous pocket in nude mice and harvested after 4 and 8 weeks. Both HA and collagen constructs promoted endochondral bone formation with vascularization and bone marrow development; however, the HA constructs fused to form integrated bone tissues and the bone marrow developed along the space between the two adhered grafts in all implanted pockets (n = 5). In the collagen constructs, the integration was observed in 40% of the pockets (n = 5). Microcomputer CT analysis revealed that the bone volume of HA constructs was larger than that of collagen constructs. In conclusion, compared to collagen hydrogels, HA hydrogels had superior potential to generate integrated bone with vascularization and bone marrow development. This study provides valuable insights for applying ECO-mediated bone tissue engineering approaches for the repair of critical-sized bone defects.

Improvement of bioactivity with dual bioceramic incorporation to nanofibrous PCL scaffolds

Bone tissue injuries, diseases or related clinical interventions require bone tissue engineering (BTE) approaches for regeneration of large bone defects, especially for compromised situations. Most BTE applications in literature focused on composites of polymers with a single type of bioceramic. However, native bone matrix has various inorganic components. Accordingly, this study aimed to investigate the use of dual bioceramics in BTE scaffolds prepared by wet-electrospinning of Poly-caprolactone (PCL) and bioceramics; hydroxyapatite (HA), carbonated hydroxyapatite (CHA) and akermanite (AKR). After physicochemical characterizations of single and dual bioceramic-discs, it was found that AKR component should be at most 10% and thus, fibrous 3D composite scaffolds (PCL/HA; PCL/CHA; PCL/90HA/10AKR; PCL/90CHA/10AKR) were prepared. Scaffolds were then compared for degradation, swelling and microstructure properties. PCL/CHA, PCL/HA/AKR and PCL/CHA/AKR scaffolds improved apatite formation and presence of CHA, AKR or both in scaffolds increased Ca/P precipitation compared to HA involving scaffolds. Specifically, when two bioceramics were introduced, there was a noticeable increase in mineralization. No significant weight change was detected for scaffolds after two weeks of incubation. Presence of bioceramics in scaffolds significantly reduced swelling of PCL scaffolds with PCL/90HA10AKR and PCL/90CHA10AKR having higher swelling results than PCL/HA and PCL/CHA, respectively. Additionally, scaffolds with dual bioceramics (90HA10AKR, 90CHA10AKR) had higher cell proliferation for bone cells (Saos-2). In terms of osteogenic activity all groups showed increased ALP activity at 14 days compared to 7 days with PCL/CHA scaffolds having the highest value. Results suggests that newly developed PCL/90HA10AKR and PCL/90CHA10AKR composites hold potential for bone tissue engineering applications.

Nanomaterials-Incorporated Chemically Modified Gelatin Methacryloyl-Based Biomedical Composites: A Novel Approach for Bone Tissue Engineering.

Gelatin methacryloyl (GelMA)-based composites are evolving three-dimensional (3D) networking hydrophilic protein composite scaffolds with high water content. These protein composites have been devoted to biomedical applications due to their unique abilities, such as flexibility, soft structure, versatility, stimuli-responsiveness, biocompatibility, biodegradability, and others. They resemble the native extracellular matrix (ECM) thanks to their remarkable cell-adhesion and matrix-metalloproteinase (MMP)-responsive amino acid motifs. These favorable properties promote cells to proliferate and inflate within GelMA-protein scaffolds. The performance of GelMA composites has been enriched using cell-amenable components, including peptides and proteins with a high affinity to harmonize cellular activities and tissue morphologies. Due to their inimitable merits, GelMA systems have been used in various fields such as drug delivery, biosensor, the food industry, biomedical, and other health sectors. The current knowledge and the role of GelMA scaffolds in bone tissue engineering are limited. The rational design and development of novel nanomaterials-incorporated GelMA-based composites with unique physicochemical and biological advantages would be used to regulate cellular functionality and bone regeneration. Substantial challenges remain. This review focuses on recent progress in mitigating those disputes. The study opens with a brief introduction to bone tissue engineering and GelMA-based composites, followed by their potential applications in bone tissue engineering. The future perspectives and current challenges of GelMA composites are demonstrated. This review would guide the researchers to design and fabricate more efficient multifunctional GelMA-based composites with improved characteristics for their practical applications in bone tissue engineering and biomedical segments.

Eco‐Sustainable Approaches in Bone Tissue Engineering: Evaluating the Angiogenic Potential of Different Poly(3‐Hydroxybutyrate‐Co‐3‐Hydroxyhexanoate)–Nanocellulose Composites with the Chorioallantoic Membrane Assay

Additive manufacturing (AM) has opened new frontiers in precision medicine, giving the possibility of fabricating patient‐customized scaffolds to replace damaged tissues. In particular, bone tissue engineering (BTE) has benefited the most from the introduction of AM. In contrast, the impelling environmental issues are moving all the industrial sectors toward a sustainable development, avoiding the use of nonrenewable resources. Since thermoplastic polymers, such as poly(lactic acid) (PLA) and polycaprolactone (PCL), are massively used in the biomedical field, the primary objective of this study is to encourage the use of biodegradable and compostable materials also in BTE. Focusing on poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) (PHBH), an eco‐friendly material with good biocompatibility and biodegradability, reinforced with cellulose nanocrystals (CNCs), 3D‐printed scaffolds through fused filament fabrication (FFF) are realized. Since vascularization is mandatory for bone regeneration processes and for a successful scaffold integration, the angiogenic potential of different PHBH–CNC formulations in vitro is tested, evaluating the colonization of the scaffolds by endothelial cells, and in ovo with the chorioallantoic membrane (CAM) assay. The final goal is to define the best geometry of the scaffold and PHBH–CNC composition that can trigger vascularization in BTE applications, with the ultimate aim of giving greater guarantees of osseointegration.

Improved Osseointegration of Selective Laser Melting Titanium Implants with Unique Dual Micro/Nano-Scale Surface Topography.

Selective laser melting manufacture of patient specific Ti implants is serving as a promising approach for bone tissue engineering. The success of implantation is governed by effective osseointegration, which depends on the surface properties of implants. To improve the bioactivity and osteogenesis, the universal surface treatment for SLM-Ti implants is to remove the primitive roughness and then reengineer new roughness by various methods. In this study, the micro-sized partially melted Ti particles on the SLM-Ti surface were preserved for assembling mesoporous bioactive glass nanospheres to obtain a unique micro/nano- topography through combination of SLM manufacture and sol-gel processes. The results of simulated body fluid immersion test showed that bioactive ions (Ca, Si) can be continuously and stably released from the MBG nanospheres. The osseointegration properties of SLM-Ti samples, examined using pre-osteoblast cells, showed enhanced adhesion and osteogenic differentiation compared with commercial pure titanium commonly used as orthopedic implants. Overall, the developed approach of construction of the dual micro/nano topography generated on the SLM-Ti native surface could be critical to enhance musculoskeletal implant performance.

An in vivo toxicity assessment of piezoelectric sodium potassium niobate [NaxK1-xNbO3 (x=0.2-0.8)] nanoparticulates towards bone tissue engineering approach.

One of the recent challenges in the design/development of prosthetic orthopedic implants is to address the concern of local/systemic toxicity of debris particles, released due to wear or degradation. Such debris particles often lead to inflammation at the implanted site or aseptic loosening of the prosthesis which results in failure of the implant during long run. Several in vitro studies demonstrated the potentiality of piezoelectric sodium potassium niobate [NaxK1-xNbO3 (x=0.2, 0.5, 0.8), NKN] as an emerging next-generation polarizable orthopedic implant. In this perspective, we performed an in vivo study to examine the local and systemic toxicity of NKN nanoparticulates, as a first report. In the present study, male Wistar rats were intra-articularly injected to the knee joint with 100μl of NKN nanoparticulates (25mg/ml in normal saline). After 7days of exposure, the histopathological analyses demonstrate the absence of any inflammation or dissemination of nanoparticulates in vital organs such as heart, liver, kidney and spleen. The anti-inflammatory cytokines (IL-4 and IL-10) profile analyses suggest the increased anti-inflammatory response in the treated rats as compared to non-injected (control) rats, preferably for the sodium and potassium rich NKN i.e., Na0.8K0.2NbO3 and Na0.2K0.8NbO3. The biochemical analyses revealed no pathological changes in the liver and kidney of particulate treated rats. The present study is the first proof to confirm the non-toxic nature of NKN nanoparticulates which provides a step forward towards the development of prosthetic orthopedic implants using biocompatible piezoelectric NKN ceramics.

Correction of large jawbone defect in the mouse using immature osteoblast-like cells and a 3D polylactic acid scaffold.

Bone tissue engineering has been developed using a combination of mesenchymal stem cells (MSCs) and calcium phosphate-based scaffolds. However, these complexes cannot regenerate large jawbone defects. To overcome this limitation of MSCs and ceramic scaffolds, a novel bone regeneration technology must be developed using cells possessing high bone forming ability and a scaffold that provides space for vertical bone augmentation. To approach this problem in our study, we developed alveolar bone-derived immature osteoblast-like cells (HAOBs), which have the bone regenerative capacity to correct a large bone defect when used as a grafting material in combination with polylactic acid fibers that organize the 3D structure and increase the strength of the scaffold material (3DPL). HAOB-3DPL constructs could not regenerate bone via xenogeneic transplantation in a micromini pig alveolar bone defect model. However, the autogenic transplantation of mouse calvaria-derived immature osteoblast-like cells (MCOBs) isolated using the identical protocol for HAOBs and mixed with 3DPL scaffolds successfully regenerated the bone in a large jawbone defect mouse model, compared to the 3DPL scaffold alone. Nanoindentation analysis indicated that the regenerated bone had a similar micromechanical strength to native bone. In addition, this MCOB-3DPL regenerated bone possesses osseointegration ability wherein a direct structural connection is established with the titanium implant surface. Hence, a complex formed between a 3DPL scaffold and immature osteoblast-like cells such as MCOBs represents a novel bone tissue engineering approach that enables the formation of vertical bone with the micromechanical properties required to treat large bone defects.

Robocasting of multicomponent sol-gel–derived silicate bioactive glass scaffolds for bone tissue engineering

Robocasting is universally recognized as an affordable and reproducible manufacturing strategy to process glass and glass-ceramic materials in the form of highly ordered porous scaffolds for bone tissue engineering (BTE) applications. Nevertheless, while being widely applied to melt-derived bioactive glasses, this technique was seldom implemented with sol-gel materials due to the intrinsic difficulties in producing suitable inks for extrusion and thus, good printing outcomes. The present experimental work describes a new and relatively easy method to manufacture multicomponent sol-gel bioactive silicate scaffolds (oxide system: 47.5SiO2–20CaO–10MgO–10Na2O-10 K2O-2.5P2O5, mol.%) using dried gels as basic material within the ink composition, which allows by-passing intermediate heat treatments that are usually detrimental to the bioactive potential of the material in physiological environment. The scaffolds, exhibiting a total porosity of 81 vol%, were characterized in terms of morphological-compositional features and bioactivity in simulated body fluid (SBF), paying special attention to ion release and surface modifications occurring upon soaking (apatite-forming ability). The compressive strength of the scaffolds (around 5 MPa) was comparable to that of human cancellous bone. Collectively, the results supported the possibility of using robocast sol-gel–derived scaffolds in BTE approaches. Furthermore, a comparison with robocast scaffolds based on a melt-derived glass with the same composition was reported in order to investigate the effect of the synthesis route on the dissolution behavior and morphological features of the final biomaterials.

Mussel-inspired polydopamine decorated alginate dialdehyde-gelatin 3D printed scaffolds for bone tissue engineering application.

This study utilized extrusion-based 3D printing technology to fabricate calcium-cross-linked alginate dialdehyde-gelatin scaffolds for bone regeneration. The surface of polymeric constructs was modified with mussel-derived polydopamine (PDA) in order to induce biomineralization, increase hydrophilicity, and enhance cell interactions. Microscopic observations revealed that the PDA layer homogeneously coated the surface and did not appear to induce any distinct change in the microstructure of the scaffolds. The PDA-functionalized scaffolds were more mechanically stable (compression strength of 0.69 ± 0.02 MPa) and hydrophilic (contact angle of 26) than non-modified scaffolds. PDA-decorated ADA-GEL scaffolds demonstrated greater durability. As result of the 18-days immersion in simulated body fluid solution, the PDA-coated scaffolds showed satisfactory biomineralization. Based on theoretical energy analysis, it was shown that the scaffolds coated with PDA interact spontaneously with osteocalcin and osteomodulin (binding energy values of −35.95 kJ mol−1 and −46.39 kJ mol−1, respectively), resulting in the formation of a protein layer on the surface, suggesting applications in bone repair. PDA-coated ADA-GEL scaffolds are capable of supporting osteosarcoma MG-63 cell adhesion, viability (140.18% after 7 days), and proliferation. In addition to increased alkaline phosphatase secretion, osteoimage intensity also increased, indicating that the scaffolds could potentially induce bone regeneration. As a consequence, the present results confirm that 3D printed PDA-coated scaffolds constitute an intriguing novel approach for bone tissue engineering.

The effect of hypergravity, hyperbaric pressure, and hypoxia on osteogenic differentiation of adipose stem cells

Human adipose stem cells (ASCs) hold great potential for regenerative medicine approaches, including osteogenic regeneration of bone defects, that fail to heal autonomously. Osteogenic differentiation of stem cells is dependent on the stimulation of biophysical factors. In the present study, the effects of hypergravity, hypoxia, and hyperbaric treatment were investigated on adipose stem cell (ASC) metabolic activity, quantified by PrestoBlue conversion, and cell numbers, evaluated by crystal violet staining. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity and cresolphthalein staining of calcium deposition. Differentiation was performed for 12 days, which was accompanied by periodical stimulation. Increasing gravity forces up to 50x g did not affect ASC viability, but it enhanced osteogenic markers with a strongest effect between 20 and 30x g. Hyperbaric stimulation at 3 bar decreased ASC cell numbers but increased ALP activity and calcium deposition. Hypoxia at 8 % atmospheric oxygen did not affect ASC proliferation, while cell numbers were reduced at 3 % oxygen. Furthermore, hypoxic conditions produced opposing results on osteogenic markers, as ALP activity increased whereas cresolphthalein staining decreased upon stimulation. These data demonstrated that intermittent short duration of basal physical or chemical impulses interfere with the osteogenic differentiation of ASCs. Our findings could be of specific relevance in ASC based therapies for regenerative medicine and bone tissue engineering approaches.

Three‐dimensional bioprinting of tragacanth/hydroxyapaptite modified alginate bioinks for bone tissue engineering with tunable printability and bioactivity

AbstractThree‐dimensional (3D) bioprinting of composite scaffolds using biodegradable natural polymers are very promising approach for bone tissue engineering. In this research, we hypothesized that the use of tragacanth gum with alginate could improve the printability and rheological properties of alginate. We showed that the blending of 1 wt% tragacanth with the 3 wt% alginate‐based bioink significantly improved the compressive strength, viscosity and printability properties, resolution, and shape fidelity of alginate bioink. In addition, the presence of tragacanth decreased the rate of degradation in alginate scaffold within 21 days. On the other hand, hydroxyapatite (HA) could support alginate as an osteoconductive component in alginate‐tragacanth composite scaffolds. HA was in situ incorporated into an alginate‐tragacanth bioink. ALP activity, calcium deposition and BMP‐2 and osteocalcin genes expression showed an increase over time indicating differentiation of MC3T3 cells into osteoblasts in the presence of HA particles than that in the alginate‐tragacanth group. In addition, the expression of CD105, CD44, and CD90, as specific surface markers, were significantly increased in differentiated osteoblasts. The in vitro results demonstrated that HA particles dispersed in the alginate‐tragacanth matrix could promote osteogenic differentiation of MC3T3 cells in a 3D‐bioprinted construct and this scaffold material could be considered to repair large bone tissue defects.

The interplay of collagen/bioactive glass nanoparticle coatings and electrical stimulation regimes distinctly enhanced osteogenic differentiation of human mesenchymal stem cells

Increasing research has incorporated bioactive glass nanoparticles (BGN) and electric field (EF) stimulation for bone tissue engineering and regeneration applications. However, their interplay and the effects of different EF stimulation regimes on osteogenic differentiation of human mesenchymal stem cells (hMSC) are less investigated. In this study, we introduced EF with negligible magnetic field strength through a well-characterized transformer-like coupling (TLC) system, and applied EF disrupted (4/4) or consecutive (12/12) regime on type I collagen (Col) coatings with/without BGN over 28 days. Additionally, dexamethasone was excluded to enable an accurate interpretation of BGN and EF in supporting osteogenic differentiation. Here, we demonstrated the influences of BGN and EF on collagen topography and maintaining coating stability. Coupled with the release profile of Si ions from the BGN, cell proliferation and calcium deposition were enhanced in the Col-BGN samples after 28 days. Further, osteogenic differentiation was initiated as early as d 7, and each EF regime was shown to activate distinct pathways. The disrupted (4/4) regime was associated with the BMP/Smad4 pathways that up-regulate Runx2/OCN gene expression on d 7, with a lesser effect on ALP activity. In contrast, the canonical Wnt/β-Catenin signaling pathway activated through mechanotransduction cues is associated with the consecutive (12/12) regime, with significantly elevated ALP activity and Sp7 gene expression reported on d 7. In summary, our results illustrated the synergistic effects of BGN and EF in different stimulation regimes on osteogenic differentiation that can be further exploited to enhance current bone tissue engineering and regeneration approaches. Statement of significanceThe unique release mechanisms of silica from bioactive glass nanoparticles (BGN) were coupled with pulsatile electric field (EF) stimulation to support hMSC osteogenic differentiation, in the absence of dexamethasone. Furthermore, the interplay with consecutive (12/12) and disrupted (4/4) stimulation regimes was investigated. The reported physical, mechanical and topographical effects of BGN and EF on the collagen coating, hMSC and the distinct progression of osteogenic differentiation (canonical Wnt/β-Catenin and BMP/Smad) triggered by respective stimulation regime were not explicitly reported previously. These results provide the fundamentals for further exploitations on BGN composites with metal ions and rotation of EF regimes to enhance osteogenic differentiation. The goal is sustaining continual osteogenic differentiation and achieving a more physiologically-relevant state and bone constructs in vitro.

A novel electrospinning method for self-assembled tree-like fibrous scaffolds: Microenvironment-associated regulation of MSC behavior and bone regeneration

• We fabricated biomimetic fibrous scaffolds via a novel electrospinning setup. • The scaffolds present fibrillar microenvironments with microfiber/nanonet networks. • The scaffolds differently regulate MSC behavior regarding to their fiber topographies. • Topography-sensitive mechanosensing enhances osteogenesis and bone regeneration. • The electrospun method provides an advanced approach for bone tissue engineering. Numerous studies highlight advantages of electrospun scaffolds in bone tissue engineering, in which cellular behavior is tightly affected by fiber topographical cues of scaffolds. However, the classic electrospinning setup limits a desired presentation of biomimetic fibrous microenvironments that sense mechanosignaling and regulate stem cell behavior. The aims of this study were to fabricate advanced as-spun scaffolds presenting tree-like microfiber/nanonet networks and to evaluate their regulatory potentials on behavior of human mesenchymal stem cells (hMSCs) and bone regeneration. Here we developed a novel electrospinning setup that allowed the presentation of patterned Trunk microfibers (TMF) and/or branched nanonet fibers (BNnFs) in biomimetic fibrous scaffolds. As the cellular mechanisms, anisotropic-hierarchical topography of TMF controlled behavior of hMSCs through focal adhesion formation and Yes-associated protein (YAP) induction, whereas BNnF disturbed such mechanosensing responses in the cells. The fiber microenvironment-related expression and nuclear localization of YAP were. also correlated with the potentials of as-spun scaffolds to enhance osteogenic differentiation of the hMSCs and alveolar bone defect healing in an animal model. Collectively, this study provides an advanced approach of the modified electrospinning setup for presentation of biomimetic fibrillar microenvironments in as-spun scaffolds along with their application in stem cell behavior regulation and regenerative tissue engineering.