Printable oxygen-generating biodegradable scaffold for thicker tissue-engineered medical products.

BACKGROUND: Due to the increasing demand to generate thick and vascularized tissue-engineered constructs, novel strategies are currently being developed. An effective example is the fabrication of a 3D scaffold containing oxygen-releasing biomaterials to solve the limitations of gas diffusion and transport within transplanted tissues or devices. METHODS: In this study, we developed a biodegradable scaffold made of polycaprolactone (PCL) mixed with oxygen-generating calcium peroxide (CPO) to design new structures for regenerative tissue using a 3D printer capable of forming arbitrarily shapes. RESULTS AND CONCLUSION: When osteoblast progenitor cells (MC3T3-E1 cells) were cultured under hypoxic conditions on scaffolds fabricated with this technique, it was shown that cell death was reduced by the new scaffolds. Therefore, the results suggest that 3D-printed scaffolds made from biodegradable oxygen-releasing materials may be useful for tissue engineering and regeneration.

Hydrostatic pressure prevents chondrocyte differentiation through heterochromatin remodeling.

Articular cartilage protects and lubricates joints for smooth motion and transmission of loads. Owing to its high water content, chondrocytes within the cartilage are exposed to high levels of hydrostatic pressure, which has been shown to promote chondrocyte identity through unknown mechanisms. Here, we investigate the effects of hydrostatic pressure on chondrocyte state and behavior, and discover that application of hydrostatic pressure promotes chondrocyte quiescence and prevents maturation towards the hypertrophic state. Mechanistically, hydrostatic pressure reduces the amount of trimethylated H3K9 (K3K9me3)-marked constitutive heterochromatin and concomitantly increases H3K27me3-marked facultative heterochromatin. Reduced levels of H3K9me3 attenuates expression of pre-hypertrophic genes, replication and transcription, thereby reducing replicative stress. Conversely, promoting replicative stress by inhibition of topoisomerase II decreases Sox9 expression, suggesting that it enhances chondrocyte maturation. Our results reveal how hydrostatic pressure triggers chromatin remodeling to impact cell fate and function.This article has an associated First Person interview with the first author of the paper.

Intracellular calcium ion transients evoked by cell poking independently of released autocrine ATP in Madin-Darby canine kidney cells.

The mechanical stimulation induced by poking cells with a glass needle activates Piezo1 receptors and the adenosine triphosphate (ATP) autocrine pathway, thus increasing intracellular Ca2+ concentration. The differences between the increase in intracellular Ca2+ concentration induced by cell poking and by ATP-only stimulation have not been investigated. In this study, we investigated the Ca2+ signaling mechanism induced by autocrine ATP release during Madin-Darby Canine Kidney cell membrane deformation by cell poking. The results suggest that the pathways for supplying Ca2+ into the cytoplasm were not identical between cell poking and conventional ATP stimulation. The functions of the G protein-coupled receptor (GPCR) subunits (G α $\alpha $ q, G ß Î³ $\beta \gamma $ ), ATP-activated receptor and the upstream Ca2+ release signal from the intracellular endoplasmic reticulum Ca2+ store, were investigated. The results show that G α $\alpha $ q plays a major role in the Ca2+ response evoked by ATP-only stimulation, while cell poking induces a Ca2+ response requiring the involvement of both G α $\alpha $ q and G ß Î³ $\beta \gamma $ units simultaneously. These results suggest that GPCR are not only activated by ATP-only stimulation or autocrine ATP release during Ca2+ signaling, but also activated by the mechanical effects of cell poking.

Local Strain Distribution and Increased Intracellular Ca2+ Signaling in Bovine Articular Cartilage Exposed to Compressive Strain.

Articular cartilage is exposed to compressive strain of approximately 10% under physiological loads in vivo, and intracellular Ca2+ signaling is one of the earliest responses in chondrocytes under this physical stimulation. However, it remains unknown whether compressive strain itself evokes intracellular Ca2+ signaling in chondrocytes located within each layer (from surface to deep) in an equal manner with physiological levels of strain. The purpose of this study, therefore, was to determine the distribution of local strain and increased intracellular Ca2+ signaling in layer-dependent cell populations in response to 10% compressive strain loading. For this purpose, the time course of strain was measured in each layer to calculate layer-specific deformation properties. In addition, layer-specific changes in chondrocyte intracellular Ca2+ signals were recorded over time using a fluorescent Ca2+ indicator, Fluo-3, to establish ratios of cells with increased Ca2+ signaling at each depth of cartilage under static conditions or exposed to compression. The results showed that the surface layer was compressed with a larger strain compared with other layers. Few cells with Ca2+ signaling were observed under static conditions. Percentages of responsive cells within compressed cartilage were higher than those within cartilage under static conditions. However, increased intracellular Ca2+ signals were observed in a prominent number of chondrocytes within the deep layer, but not the surface layer, of compressed cartilage. Our results suggest that at a physiological compression level, Ca2+ is upregulated, but the stimulation of Ca2+ signaling in articular cartilage is not simply defined by local deformation.

Enhancing osteogenic differentiation of MC3T3-E1 cells by immobilizing inorganic polyphosphate onto hyaluronic acid hydrogel.

In tissue engineering, precise control of cues in the microenvironment is essential to stimulate cells to undergo bioactivities such as proliferation, differentiation, and matrix production. However, current approaches are inefficient in providing nondepleting cues. In this study, we have developed a novel bioactive hydrogel (HAX-PolyP) capable of enhancing tissue growth by conjugating inorganic polyphosphate chains onto hyaluronic acid macromers. The immobilized polyphosphates provided constant osteoconductive stimulation to the embedded murine osteoblast precursor cells, resulting in up-regulation of osteogenic marker genes and enhanced levels of ALP activity. The osteoconductive activity was significantly higher when compared to those stimulated with free-floating polyphosphates. Even at very low concentrations, immobilization of polyphosphates onto the scaffold allowed sufficient signaling leading to more effective osteoconduction. These results demonstrate the potential of our novel material as an injectable bioactive scaffold, which can be clinically useful for developing bone grafts and bone regeneration applications.

Bone regeneration in calvarial defects in a rat model by implantation of human bone marrow-derived mesenchymal stromal cell spheroids.

Mesenchymal stem cell (MSC) condensation contributes to membrane ossification by enhancing their osteodifferentiation. We investigated bone regeneration in rats using the human bone marrow-derived MSC-spheroids prepared by rotation culture, without synthetic or exogenous biomaterials. Bilateral calvarial defects (8 mm) were created in nude male rats; the left-sided defects were implanted with MSC-spheroids, ß-tricalcium phosphate (ß-TCP) granules, or ß-TCP granules + MSC-spheroids, while the right-sided defects served as internal controls. Micro-computed tomography and immunohistochemical staining for osteocalcin/osteopontin indicated formation of new, full-thickness bones at the implantation sites, but not at the control sites in the MSC-spheroid group. Raman spectroscopy revealed similarity in the spectral properties of the repaired bone and native calvarial bone. Mechanical performance of the bones in the MSC-implanted group was good (50 and 60% those of native bones, respectively). All tests showed poor bone regeneration in the ß-TCP and ß-TCP + MSC-spheroid groups. Thus, significant bone regeneration was achieved with MSC-spheroid implantation into bone defects, justifying further investigation.

Differentiation-inducing effect of osteoclast microgrooves for the purpose of three-dimensional design of regenerated bone.

In vivo bone remodeling is promoted by the balance between osteoclast and osteoblast activity. Conventional research on bone regeneration has mainly focused on increasing osteoblast activity, with limited studies on the effects of scaffold topography on cell differentiation. Here, we examined the effect of microgroove-patterned substrate with spacings ranging from 1 to 10 µm on the differentiation of rat bone marrow-derived osteoclast precursors. Tartrate-resistant acid phosphatase (TRAP) staining and relative gene expression quantification showed that osteoclast differentiation was enhanced in substrate with 1 µm microgroove spacing compared with that in the other groups. Additionally, the ratio of podosome maturation stages in substrate with 1 µm microgroove spacing exhibited a distinct pattern, which was characterized by an increase in the ratio of belts and rings and a decrease in that of clusters. However, myosin II abolished the effects of topography on osteoclast differentiation. Overall, these showed that the reduction of myosin II tension in the podosome core by an integrin vertical vector increased podosome stability and promoted osteoclast differentiation in substrates with 1 µm microgroove spacing, including that microgroove design plays an important role in scaffolds for bone regeneration. STATEMENT OF SIGNIFICANCE: Reduction of myosin II tension in the podosome core, facilitated by an integrin vertical vector, resulted in an enhanced osteoclast differentiation, concomitant with an increase in podosome stability within 1-µm-spaced microgrooves. These findings are anticipated to serve as valuable indicators for the regulation of osteoclast differentiation through the manipulation of biomaterial surface topography in tissue engineering. Furthermore, this study contributes to the lucidation of the underlying mechanisms governing cellular differentiation by providing insights into the impact of the microtopographical environment.

Effect of Pressure Conditions in Uterine Decellularization Using Hydrostatic Pressure on Structural Protein Preservation.

Uterine regeneration using decellularization scaffolds provides a novel treatment for uterine factor infertility. Decellularized scaffolds require maximal removal of cellular components and minimal damage to the extracellular matrix (ECM). Among many decellularization methods, the hydrostatic pressure (HP) method stands out due to its low cytotoxicity and superior ECM preservation compared to the traditional detergent methods. Conventionally, 980 MPa was utilized in HP decellularization, including the first successful implementation of uterine decellularization previously reported by our team. However, structural protein denaturation caused by exceeding pressure led to a limited regeneration outcome in our previous research. This factor urged the study on the effects of pressure conditions in HP methods on decellularized scaffolds. The authors, therefore, fabricated a decellularized uterine scaffold at varying pressure conditions and evaluated the scaffold qualities from the perspective of cell removal and ECM preservation. The results show that by using lower decellularization pressure conditions of 250 MPa, uterine tissue can be decellularized with more preserved structural protein and mechanical properties, which is considered to be promising for decellularized uterine scaffold fabrication applications.

Simultaneous Hydrostatic and Compressive Loading System for Mimicking the Mechanical Environment of Living Cartilage Tissue.

In vivo, articular cartilage tissue is surrounded by a cartilage membrane, and hydrostatic pressure (HP) and compressive strain increase simultaneously with the compressive stress. However, it has been impossible to investigate the effects of simultaneous loading in vitro. In this study, a bioreactor capable of applying compressive stress under HP was developed to reproduce ex vivo the same physical loading environment found in cartilage. First, a HP stimulation unit was constructed to apply a cyclic HP pressure-resistant chamber by controlling a pump and valve. A compression-loading mechanism that can apply compressive stress using an electromagnetic force was implemented in the chamber. The synchronization between the compression and HP units was evaluated, and the stimulation parameters were quantitatively evaluated. Physiological HP and compressive strain were applied to the chondrocytes encapsulated in alginate and gelatin gels after applying high HP at 25 MPa, which induced damage to the chondrocytes. It was found that compressive stimulation increased the expression of genes related to osteoarthritis. Furthermore, the simultaneous application of compressive strain and HP, which is similar to the physiological environment in cartilage, had an inhibitory effect on the expression of genes related to osteoarthritis. HP alone also suppressed the expression of osteoarthritis-related genes. Therefore, the simultaneous hydrostatic and compressive stress-loading device developed to simulate the mechanical environment in vivo may be an important tool for elucidating the mechanisms of disease onset and homeostasis in cartilage.

Modulation of the long non-coding RNA Mir155hg by high, but not moderate, hydrostatic pressure in cartilage precursor cells.

Osteoarthritis (OA) is the most common joint disease in older adults and is characterized by a gradual degradation of articular cartilage due to decreased cartilage matrix gene expression and increased expression of genes involved in protein degradation, apoptosis and inflammation. Due to the high water content of cartilage, one of the main physical stimuli sensed by chondrocytes is hydrostatic pressure. We previously showed that high pressure above 20 MPa induced gene expression changes in chondrocyte precursor cells similar to what is observed in OA. Micro-RNAs are small non-coding RNAs essential to many physiological and pathological process including OA. As the micro-RNA miR-155 has been found increased in OA chondrocytes, we investigated the effects of high pressure on the expression of the miR-155 host gene Mir155hg. The chondrocyte progenitor cell line ATDC5 was pressurized under hydrostatic pressure up to 25 MPa and the expression of Mir155hg or the resulting micro-RNAs were measured; pharmacological inhibitors were used to identify the signaling pathways involved in the regulation of Mir155hg. We found that Mir155hg is strongly and rapidly up-regulated by high, but not moderate, pressure in chondrocyte progenitor cells. This up-regulation likely involves the membrane channel pannexin-1 and several intracellular signaling molecules including PKC and Src. MiR-155-5p and -3p were also up-regulated by pressure though somewhat later than Mir155hg, and a set of known miR-155-5p target genes, including Ikbke, Smarca4 and Ywhae, was affected by pressure, suggesting that Mir155hg may have important roles in cartilage physiology.

Internal radial perfusion bioreactor promotes decellularization and recellularization of rat uterine tissue.

The advances in infertility treatment technologies such as in vitro fertilization (IVF) help many infertile women to be able to get pregnant. However, these infertility treatments cannot be applied to women who are suffering from absolute uterine factor. Fabrication of functional scaffold in tissue engineering approach is believed to play an important role for uterine regeneration and uterus replacement for treating absolute uterine factor infertility. In this research, we developed an internal radial perfusion bioreactor to promote decellularization and recellularization for fabrication of functional engineered uterine tissue. As a result, the DNA contents of the decellularized uterine tissue with high hydrostatic pressure followed by 7 days internal perfusion washing decreased by 90% compared to native tissue. Collagen and proteoglycan contents in the pressurized uterine tissue with the internal perfusion bioreactor, static (control) and shaking treatment with high hydrostatic pressure showed no significant change compared to the native tissue. The newly developed perfusion bioreactor also enabled to recellularize in the decellularized tissue with statistically significant increase of DNA by 614% compared to non-seeded cell groups. Vimentin and 4',6-diamidino-2-phenylindole (DAPI) was homogeneously expressed in the seeded endometrial stromal cells in the recellularized tissue fabricated using the bioreactor. With the developed internal radial perfusion bioreactor, we are the first group to successfully recellularized uterine tissue in all layers including epithelium, endometrium and myometrium. These results showed that the internal perfusion bioreactor has potential to be utilized for fabrication of functional engineered tissue to promote tissue regeneration.

Quantitative analysis of human platelet adhesions under a small-scale flow device.

To realize real-time evaluation of human platelet adhesions onto material surfaces with small volumes of human platelet suspensions, we developed an apparatus consisting of a modified cone and plate-type viscometer, combined with an upright epi-fluorescence microscope. The apparatus allowed real-time evaluation of platelet-material interactions and the initial event of thrombus formation, using small platelet suspension volumes (7.5 microL) under shear flow conditions. To study the dynamic behavior of platelet-material interaction, we chose five representative opaque and transparent materials: acrylate resin (AC), polytetrafluoroethylene (PTFE), polyvynylchrolide (PVC), glass, and a monolayer of human normal umbilical cord vein endothelial cells (EC) on glass under shear flow conditions. The values of adhesiveness of human platelets to the test materials in descending order were as follows: AC > PTFE > PVC > glass > human EC. Under this new small-scale flow system, we could obtain highly reproducible data, which were comparable with results from a previously developed large-scale flow system. Therefore, the newly developed cone and plate-type rheometer is a useful instrument for testing and screening materials, and allows precise quantitative evaluation of human platelet adhesion.

New microvascular anastomotic device for end-to-side anastomosis using negative pressure; a preliminary study.

We previously developed a device for end-to-end anastomosis powered by negative pressure and demonstrated that using the device allow the operator to anastomose semi-automatically with little stress. Here, we sought to build a device for and demonstrate that negative pressure can also be used in end-to-side anastomosis which is clinically popular as end-to-end anastomosis through animal experiment using rats.The devices were constructed with a laser lithographic/3D-printing machine. Nine SD rats were used. Each of the nine rats underwent end-to-side anastomosis between the superficial epigastric vein and the femoral vein using the device. Rat was anesthetized one week later and the anastomotic site was inspected through operative microscope for patency. The anastomotic site was harvested with the device and the rat was euthanized. The anastomotic site was embedded in epon, sectioned, stained with toluidine blue, and analyzed with light microscopy. Eight of the nine anastomoses were patent immediately after the procedures, and two of the nine were patent at 1 week after the procedures. In the failed cases, the vessels dislocated from the device because the clamps loosened during the observation period after the operation. The experiments have shown that the device using negative pressure can also be applied to end-to-side microvascular anastomosis. The patency rate is low and further improvement is required.

Layer dependence in strain distribution and chondrocyte damage in porcine articular cartilage exposed to excessive compressive stress loading.

Exposure to excessive stress is associated with the pathogenesis of osteoarthritis, a joint disease involved in the degeneration of articular cartilage. Mechanical properties of mature articular cartilage are known to be depth zone-dependent. Although chondrocyte death was observed in articular cartilage after excessive stress loading in vitro, few studies have investigated the correlation between chondrocyte death and local mechanical strains in a depth dependent manner. Here, we developed a real-time observation system of cut cartilage samples under an excessive stress loading (18 MPa) at low (3.5%/s) and high (35%/s) strain rates on the microscope stage, which is regarded as injurious compression in vivo. Using this system, real-time monitoring of local deformations was conducted during compression, and local chondrocyte death was investigated after short-term culture. The results showed that the dead cells were mainly observed in the surface layer at a high strain rate. In contrast, the dead cells were relatively concentrated not in the surface layer but in the middle layer at a low strain rate. The local strain measurements showed that the dead cell distributions were correlated with depth-dependent local strain rates at both low and high strain rates. Moreover, when the surface layer was removed, both depth-dependence in dead cell distributions and in local strain rates disappeared at low and high strain rates. Although the mechanisms underlying mechanically induced osteoarthritis are still elusive, those results suggest a correlation between local chondrocyte death and transient strain rates in a depth dependent manner, and the surface layer played a crucial role in regulating chondrocyte damages and local strains in middle and deep layers. Our study, therefore, could contribute to an analytical understanding of cartilage degeneration under excessive stress loadings.

3D culture of osteoblast-like cells by unidirectional or oscillatory flow for bone tissue engineering.

A medium perfusion system is expected to be beneficial for three-dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast-like MC 3T3-E1 cells were 3D-cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell-seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering.

Assessment of the Inner Surface Microstructure of Decellularized Cortical Bone by a Scanning Electron Microscope.

The microstructural changes of bones, which form a hierarchy of skeletal tissue, vary, depending on their condition, and are affected by the behaviors of bone cells. The purpose of this study is to assess the microstructural changes in the inner femoral surface of Sprague Dawley rats according to the conditions using a scanning electron microscope. Microstructural differences on the endocortical surface were observed in the characteristics of osteocytic canaliculi, bone fibers, and surface roughness, showing a rougher surface in old adults and an osteoporosis model by quantitative comparison. These results could be helpful for developing a basic understanding of the microstructural changes that occur on the bone surface under various conditions.

Scaffold-free cartilage by rotational culture for tissue engineering.

Our objective was to investigate the hypothesis that tissue-engineered cartilage with promising biochemical, mechanical properties can be formed by loading mechanical stress under existing cell-cell interactions analogous to those that occur in condensation during embryonic development. By loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions, we could first form a scaffold-free cartilage tissue with arbitrary shapes and a large size with promising biological, mechanical properties. The cartilage tissue which constituted of chondrocytes and ECM produced by inoculated dedifferentiated chondrocytes to a high porous simple mold has arbitrary shapes, and did not need any biodegradable scaffold to control the shape. In contrast, scaffold-free cartilage tissue cultured under static conditions could not keep their shapes; it was fragile tissue. The possibility of scaffold-free organ design was suggested because the cartilage tissue increases steadily in size with culture time; indeed, the growth of cartilage tissue starting from an arbitrary shape might be predictable by mathematical expression. For tissue-engineered cartilage formation with arbitrary shapes, biochemical and mechanical properties, loading dedifferentiated chondrocytes with mechanical stress under existing cell-cell interactions has prominent effects. Therefore, our scaffold-free cartilage model loaded mechanical stress based on a simple mold system may be applicable for tissue-engineered cartilage.

Hypergravity down-regulates c-fos gene expression via ROCK/Rho-GTP and the PI3K signaling pathway in murine ATDC5 chondroprogenitor cells.

Chondrocytes are known to be physiologically loaded with diverse physical factors such as compressive stress, shear stress and hydrostatic pressure. Although the effects of those mechanical stimuli onto various cell models have been widely studied, those of hypergravity have not yet been revealed clearly. Hereby, we hypothesized that the hypergravity affects relative positions of intracellular elements including nucleus and cytoskeletons due to their density differences, triggering mechanotransduction in the cell. The aim of this study was to investigate the effect of hypergravity on c-fos expression in the murine ATDC5 chondroprogenitor cells, as c-fos is a well known key regulator of cell proliferation and differentiation, including in chondrocytes. We first found that hypergravity down-regulated c-fos expression transiently via ROCK/Rho-GTP and PI3K signaling, and the down-regulation was suppressed by inhibition of actin polymerization.

High hydrostatic pressure induces pro-osteoarthritic changes in cartilage precursor cells: A transcriptome analysis.

Due to the high water content of cartilage, hydrostatic pressure is likely one of the main physical stimuli sensed by chondrocytes. Whereas, in the physiological range (0 to around 10 MPa), hydrostatic pressure exerts mostly pro-chondrogenic effects in chondrocyte models, excessive pressures have been reported to induce detrimental effects on cartilage, such as increased apoptosis and inflammation, and decreased cartilage marker expression. Though some genes modulated by high pressure have been identified, the effects of high pressure on the global gene expression pattern have still not been investigated. In this study, using microarray technology and real-time PCR validation, we analyzed the transcriptome of ATDC5 chondrocyte progenitors submitted to a continuous pressure of 25 MPa for up to 24 h. Several hundreds of genes were found to be modulated by pressure, including some not previously known to be mechano-sensitive. High pressure markedly increased the expression of stress-related genes, apoptosis-related genes and decreased that of cartilage matrix genes. Furthermore, a large set of genes involved in the progression of osteoarthritis were also induced by high pressure, suggesting that hydrostatic pressure could partly mimic in vitro some of the genetic alterations occurring in osteoarthritis.

An angiogenesis platform using a cubic artificial eggshell with patterned blood vessels on chicken chorioallantoic membrane.

The chorioallantoic membrane (CAM) containing tiny blood vessels is an alternative to large animals for studies involving angiogenesis and tissue engineering. However, there is no technique to design the direction of growing blood vessels on the CAM at the microscale level for tissue engineering experiments. Here, a methodology is provided to direct blood vessel formation on the surface of a three-dimensional egg yolk using a cubic artificial eggshell with six functionalized membranes. A structure on the lateral side of the eggshell containing a straight channel and an interlinked chamber was designed, and the direction and formation area of blood vessels with blood flow was artfully defined by channels with widths of 70-2000 µm, without sharply reducing embryo viability. The relationship between the size of interlinked chamber and the induction of blood vessels was investigated to establish a theory of design. Role of negative and positive pressure in the induction of CAM with blood vessels was investigated, and air pressure change in the culture chamber was measured to demonstrate the mechanism for blood vessel induction. Histological evaluation showed that components of CAM including chorionic membrane and blood vessels were induced into the channels. Based on our design theory, blood vessels were induced into arrayed channels, and channel-specific injection and screening were realized, which demonstrated proposed applications. The platform with position- and space-controlled blood vessels is therefore a powerful tool for biomedical research, which may afford exciting applications in studies involved in local stimulation of blood vessel networks and those necessary to establish a living system with blood flow from a beating heart.