Osteogenic human MSC-derived extracellular vesicles regulate MSC activity and osteogenic differentiation and promote bone regeneration in a rat calvarial defect model.

BACKGROUND: There is growing evidence that extracellular vesicles (EVs) play a crucial role in the paracrine mechanisms of transplanted human mesenchymal stem cells (hMSCs). Little is known, however, about the influence of microenvironmental stimuli on the osteogenic effects of EVs. This study aimed to investigate the properties and functions of EVs derived from undifferentiated hMSC (Naïve-EVs) and hMSC during the early stage of osteogenesis (Osteo-EVs). A further aim was to assess the osteoinductive potential of Osteo-EVs for bone regeneration in rat calvarial defects. METHODS: EVs from both groups were isolated using size-exclusion chromatography and characterized by size distribution, morphology, flow cytometry analysis and proteome profiling. The effects of EVs (10 µg/ml) on the proliferation, migration, and osteogenic differentiation of cultured hMSC were evaluated. Osteo-EVs (50 µg) or serum-free medium (SFM, control) were combined with collagen membrane scaffold (MEM) to repair critical-sized calvarial bone defects in male Lewis rats and the efficacy was assessed using µCT, histology and histomorphometry. RESULTS: Although Osteo- and Naïve-EVs have similar characteristics, proteomic analysis revealed an enrichment of bone-related proteins in Osteo-EVs. Both groups enhance cultured hMSC proliferation and migration, but Osteo-EVs demonstrate greater efficacy in promoting in vitro osteogenic differentiation, as evidenced by increased expression of osteogenesis-related genes, and higher calcium deposition. In rat calvarial defects, MEM with Osteo-EVs led to greater and more consistent bone regeneration than MEM loaded with SFM. CONCLUSIONS: This study discloses differences in the protein profile and functional effects of EVs obtained from naïve hMSC and hMSC during the early stage of osteogenesis, using different methods. The significant protein profile and cellular function of EVs derived from hMSC during the early stage of osteogenesis were further verified by a calvarial bone defect model, emphasizing the importance of using differentiated MSC to produce EVs for bone therapeutics.

Translation of biophysical environment in bone into dynamic cell culture under flow for bone tissue engineering.

Bone is a dynamic environment where osteocytes, osteoblasts, and mesenchymal stem/progenitor cells perceive mechanical cues and regulate bone metabolism accordingly. In particular, interstitial fluid flow in bone and bone marrow serves as a primary biophysical stimulus, which regulates the growth and fate of the cellular components of bone. The processes of mechano-sensory and -transduction towards bone formation have been well studied mainly in vivo as well as in two-dimensional (2D) dynamic cell culture platforms, which elucidated mechanically induced osteogenesis starting with anabolic responses, such as production of nitrogen oxide and prostaglandins followed by the activation of canonical Wnt signaling, upon mechanosensation. The knowledge has been now translated into regenerative medicine, particularly into the field of bone tissue engineering, where multipotent stem cells are combined with three-dimensional (3D) scaffolding biomaterials to produce transplantable constructs for bone regeneration. In the presence of 3D scaffolds, the importance of suitable dynamic cell culture platforms increases further not only to improve mass transfer inside the scaffolds but to provide appropriate biophysical cues to guide cell fate. In principle, the concept of dynamic cell culture platforms is rooted to bone mechanobiology. Therefore, this review primarily focuses on biophysical environment in bone and its translation into dynamic cell culture platforms commonly used for 2D and 3D cell expansion, including their advancement, challenges, and future perspectives. Additionally, it provides the literature review of recent empirical studies using 2D and 3D flow-based dynamic cell culture systems for bone tissue engineering.

Unique osteogenic profile of bone marrow stem cells stimulated in perfusion bioreactor is Rho-ROCK-mediated contractility dependent.

The fate determination of bone marrow mesenchymal stem/stromal cells (BMSC) is tightly regulated by mechanical cues, including fluid shear stress. Knowledge of mechanobiology in 2D culture has allowed researchers in bone tissue engineering to develop 3D dynamic culture systems with the potential for clinical translation in which the fate and growth of BMSC are mechanically controlled. However, due to the complexity of 3D dynamic cell culture compared to the 2D counterpart, the mechanisms of cell regulation in the dynamic environment remain relatively undescribed. In the present study, we analyzed the cytoskeletal modulation and osteogenic profiles of BMSC under fluid stimuli in a 3D culture condition using a perfusion bioreactor. BMSC subjected to fluid shear stress (mean 1.56 mPa) showed increased actomyosin contractility, accompanied by the upregulation of mechanoreceptors, focal adhesions, and Rho GTPase-mediated signaling molecules. Osteogenic gene expression profiling revealed that fluid shear stress promoted the expression of osteogenic markers differently from chemically induced osteogenesis. Osteogenic marker mRNA expression, type 1 collagen formation, ALP activity, and mineralization were promoted in the dynamic condition, even in the absence of chemical supplementation. The inhibition of cell contractility under flow by Rhosin chloride, Y27632, MLCK inhibitor peptide-18, or Blebbistatin revealed that actomyosin contractility was required for maintaining the proliferative status and mechanically induced osteogenic differentiation in the dynamic culture. The study highlights the cytoskeletal response and unique osteogenic profile of BMSC in this type of dynamic cell culture, stepping toward the clinical translation of mechanically stimulated BMCS for bone regeneration.

Hybrid material based on hyaluronan hydrogels and poly(l-lactide-co-1,3-trimethylene carbonate) scaffolds toward a cell-instructive microenvironment with long-term in vivo degradability.

Degradable polyester-based scaffolds are ideal for tissue engineering applications where long-term structural integrity and mechanical support are a requisite. However, their hydrophobic and unfunctionalized surfaces restrain their tissue-mimetic quality. Instead, hyaluronan (HA) hydrogels are able to act as cell-instructive materials with the ability to recapitulate native tissue, although HA is rapidly metabolized in vivo. Taking advantage of these distinctly diverse material properties, a degradable and concurrent hybrid hydrogel material was developed that combines the short-term tissue-relevant properties of bio-orthogonal crosslinked HA with the long-term structural and mechanical support of poly(l-lactide-co-trimethylene carbonate) (PLATMC) scaffolds. This method rendered the formulation of transparent, minimally swelling hydrogel compartments with a desirable cell-instructive "local" elastic modulus within the scaffold matrix without impeding key material properties of PLATMC. Long-term degradability over 180 days in vivo was realized by the integral PLATMC scaffold architecture obtained through either extrusion-based 3D printing or salt-particulate leaching. Intrinsic diffusion capacity within the hydrogel elicited unaffected degradation kinetics of PLATMC in vivo, despite its autocatalytic bulk degradation characteristics displayed when 3D-printed. The effect of the processing method on the material properties of PLATMC markedly extends to its in vivo degradation characteristics, and essential uniform degradation behavior can be advanced using salt-particulate leaching. Regardless of the scaffold fabrication method, the polymer exhibited a soft and flexible nature throughout the degradation period, governed by the rubbery state of the polymer. Our results demonstrate that the physicochemical properties of the hybrid hydrogel scaffold endow it with the potential to act as a cell instructive microenvironment while not affecting key material properties of PLATMC postprocessing. Importantly, the HA hydrogel does not adversely impact the degradation behavior of PLATMC, a vital aspect in the fabrication of tissue engineering constructs. The results presented herein open new avenues for the adoption of concurrent and well-defined tissue-relevant materials exhibiting the potential to recreate microenvironments for cell encapsulation and drug delivery in vivo while providing essential structural integrity and long-term degradability.

Optimization and Validation of a Custom-Designed Perfusion Bioreactor for Bone Tissue Engineering: Flow Assessment and Optimal Culture Environmental Conditions.

Various perfusion bioreactor systems have been designed to improve cell culture with three-dimensional porous scaffolds, and there is some evidence that fluid force improves the osteogenic commitment of the progenitors. However, because of the unique design concept and operational configuration of each study, the experimental setups of perfusion bioreactor systems are not always compatible with other systems. To reconcile results from different systems, the thorough optimization and validation of experimental configuration are required in each system. In this study, optimal experimental conditions for a perfusion bioreactor were explored in three steps. First, an in silico modeling was performed using a scaffold geometry obtained by microCT and an expedient geometry parameterized with porosity and permeability to assess the accuracy of calculated fluid shear stress and computational time. Then, environmental factors for cell culture were optimized, including the volume of the medium, bubble suppression, and medium evaporation. Further, by combining the findings, it was possible to determine the optimal flow rate at which cell growth was supported while osteogenic differentiation was triggered. Here, we demonstrated that fluid shear stress up to 15 mPa was sufficient to induce osteogenesis, but cell growth was severely impacted by the volume of perfused medium, the presence of air bubbles, and medium evaporation, all of which are common concerns in perfusion bioreactor systems. This study emphasizes the necessity of optimization of experimental variables, which may often be underreported or overlooked, and indicates steps which can be taken to address issues common to perfusion bioreactors for bone tissue engineering.

Efficacy of treating segmental bone defects through endochondral ossification: 3D printed designs and bone metabolic activities.

Three-dimensional printing (3D printing) is a promising technique for producing scaffolds for bone tissue engineering applications. Porous scaffolds can be printed directly, and the design, shape and porosity can be controlled. 3D synthetic biodegradable polymeric scaffolds intended for in situ bone regeneration must meet stringent criteria, primarily appropriate mechanical properties, good 3D design, adequate biocompatibility and the ability to enhance bone formation. In this study, healing of critical-sized (5 â€‹mm) femur defects of rats was enhanced by implanting two different designs of 3D printed poly(l-lactide-co-ε-caprolactone) (poly(LA-co-CL)) scaffolds seeded with rat bone marrow mesenchymal stem cells (rBMSC), which had been pre-differentiated in vitro into cartilage-forming chondrocytes. Depending on the design, the scaffolds had an interconnected porous structure of 300-500 â€‹µm and porosity of 50-65%. According to a computational simulation, the internal force distribution was consistent with scaffold designs and comparable between the two designs. Moreover, the defects treated with 3D-printed scaffolds seeded with chondrocyte-like cells exhibited significantly increased bone formation up to 15 weeks compared with empty defects. In all experimental animals, bone metabolic activity was monitored by positron emission tomography 1, 3, 5, 7, 11 and 14 weeks after surgery. This demonstrated a time-dependent relationship between scaffold design and metabolic activity. This confirmed that successful regeneration was highly reproducible. The in vitro and in vivo data indicated that the experimental setups had promising outcomes and could facilitate new bone formation through endochondral ossification.

Scaffolds in Periodontal Regenerative Treatment.

Successful periodontal regeneration requires the hierarchical reorganization of multiple tissues including periodontal ligament, cementum, alveolar bone, and gingiva. The limitation of conventional regenerative therapies has been attracting research interest in tissue engineering-based periodontal therapies where progenitor cells, scaffolds, and bioactive molecules are delivered. Scaffolds offer not only structural support but also provide geometrical clue to guide cell fate. Additionally, functionalization improves bioactive properties to the scaffold. Various scaffold designs have been proposed for periodontal regeneration. These include the fabrication of biomimetic periodontal extracellular matrix, multiphasic scaffolds with tissue-specific layers, and personalized 3D printed scaffolds. This review summarizes the basic concept as well as the recent advancement of scaffold designing and fabrication for periodontal regeneration and provides an insight of future clinical translation.

Induction of osteogenic differentiation of bone marrow stromal cells on 3D polyester-based scaffolds solely by subphysiological fluidic stimulation in a laminar flow bioreactor.

The fatal determination of bone marrow mesenchymal stem/stromal cells (BMSC) is closely associated with mechano-environmental factors in addition to biochemical clues. The aim of this study was to induce osteogenesis in the absence of chemical stimuli using a custom-designed laminar flow bioreactor. BMSC were seeded onto synthetic microporous scaffolds and subjected to the subphysiological level of fluid flow for up to 21 days. During the perfusion, cell proliferation was significantly inhibited. There were also morphological changes, with F-actin polymerisation and upregulation of ROCK1. Notably, in BMSC subjected to flow, mRNA expression of osteogenic markers was significantly upregulated and RUNX2 was localised in the nuclei. Further, under perfusion, there was greater deposition of collagen type 1 and calcium onto the scaffolds. The results confirm that an appropriate level of fluid stimuli preconditions BMSC towards the osteoblastic lineage on 3D scaffolds in the absence of chemical stimulation, which highlights the utility of flow bioreactors in bone tissue engineering.

Surface activation with oxygen plasma promotes osteogenesis with enhanced extracellular matrix formation in three-dimensional microporous scaffolds.

Various types of synthetic polyesters have been developed as biomaterials for tissue engineering. These materials commonly possess biodegradability, biocompatibility, and formability, which are preferable properties for bone regeneration. The major challenge of using synthetic polyesters is the result of low cell affinity due to their hydrophobic nature, which hinders efficient cell seeding and active cell dynamics. To improve wettability, plasma treatment is widely used in industry. Here, we performed surface activation with oxygen plasma to hydrophobic copolymers, poly(l-lactide-co-trimethylene carbonate), which were shaped in 2D films and 3D microporous scaffolds, and then we evaluated the resulting surface properties and the cellular responses of rat bone marrow stem cells (rBMSC) to the material. Using scanning electron microscopy and Fourier-transform infrared spectroscopy, we demonstrated that short-term plasma treatment increased nanotopographical surface roughness and wettability with minimal change in surface chemistry. On treated surfaces, initial cell adhesion and elongation were significantly promoted, and seeding efficiency was improved. In an osteoinductive environment, rBMSC on plasma-treated scaffolds exhibited accelerated osteogenic differentiation with osteogenic markers including RUNX2, osterix, bone sialoprotein, and osteocalcin upregulated, and a greater amount of collagen matrix and mineral deposition were found. This study shows the utility of plasma surface activation for polymeric scaffolds in bone tissue engineering.

Bone-Forming Effect of a Static Magnetic Field in Rabbit Femurs.

This study investigated the level of magnetic energy around implants possessing a static magnetic field (SMF) and assessed the in vivo influence of SMF on bone regeneration. Implants possessing a sintered neodymium magnet internally were placed in a rabbit femur. An implant without SMF was placed as control. After 12 weeks of healing in vivo, the bone samples were subjected to histologic/histomorphometric evaluation. The bone-to-implant contact for the test group and the control group were 32.4 ± 13.6% and 17.1 ± 4.5%, respectively, and the differences were statistically significant (P < .05). The results suggested that the SMF promoted new bone apposition.

Structural origin of the anisotropic and isotropic thermal expansion of K2NiF4-Type LaSrAlO4 and Sr2TiO4.

K2NiF4-type LaSrAlO4 and Sr2TiO4 exhibit anisotropic and isotropic thermal expansion, respectively; however, their structural origin is unknown. To address this unresolved issue, the crystal structure and thermal expansion of LaSrAlO4 and Sr2TiO4 have been investigated through high-temperature neutron and synchrotron X-ray powder diffraction experiments and ab initio electronic calculations. The thermal expansion coefficient (TEC) along the c-axis (αc) being higher than that along the a-axis (αa) of LaSrAlO4 [αc = 1.882(4)αa] is mainly ascribed to the TEC of the interatomic distance between Al and apical oxygen O2 α(Al-O2) being higher than that between Al and equatorial oxygen O1 α(Al-O1) [α(Al-O2) = 2.41(18)α(Al-O1)]. The higher α(Al-O2) is attributed to the Al-O2 bond being longer and weaker than the Al-O1 bond. Thus, the minimum electron density and bond valence of the Al-O2 bond are lower than those of the Al-O1 bond. For Sr2TiO4, the Ti-O2 interatomic distance, d(Ti-O2), is equal to that of Ti-O1, d(Ti-O1) [d(Ti-O2) = 1.0194(15)d(Ti-O1)], relative to LaSrAlO4 [d(Al-O2) = 1.0932(9)d(Al-O1)]. Therefore, the bond valence and minimum electron density of the Ti-O2 bond are nearly equal to those of the Ti-O1 bond, leading to isotropic thermal expansion of Sr2TiO4 than LaSrAlO4. These results indicate that the anisotropic thermal expansion of K2NiF4-type oxides, A2BO4, is strongly influenced by the anisotropy of B-O chemical bonds. The present study suggests that due to the higher ratio of interatomic distance d(B-O2)/d(B-O1) of A2(2.5+)B(3+)O4 compared with A2(2+)B(4+)O4, A2(2.5+)B(3+)O4 compounds have higher α(B-O2), and A2(2+)B(4+)O4 materials exhibit smaller α(B-O2), leading to the anisotropic thermal expansion of A2(2.5+)B(3+)O4 and isotropic thermal expansion of A2(2+)B(4+)O4. The "true" thermal expansion without the chemical expansion of A2BO4 is higher than that of ABO3 with a similar composition.

Genetic diversity of symbiotic cyanobacteria in Cycas revoluta (Cycadaceae).

The diversity of cyanobacterial species within the coralloid roots of an individual and populations of Cycas revoluta was investigated based on 16S rRNA gene sequences. Sixty-six coralloid roots were collected from nine natural populations of cycads on Kyushu and the Ryukyu Islands, covering the entire distribution range of the species. Approximately 400 bp of the 5'-end of 16S rRNA genes was amplified, and each was identified by denaturing gradient gel electrophoresis. Most coralloid roots harbored only one cyanobiont, Nostoc, whereas some contained two or three, representing cyanobiont diversity within a single coralloid root isolated from a natural habitat. Genotypes of Nostoc within a natural population were occasionally highly diverged and lacked DNA sequence similarity, implying genetic divergence of Nostoc. On the other hand, Nostoc genotypes showed no phylogeographic structure across the distribution range, while host cycads exhibited distinct north-south differentiation. Cycads may exist in symbiosis with either single or multiple Nostoc strains in natural soil habitats.