Journal of tissue science & engineering最新文献

Biochemical analysis of human brain tissue is typically done by homogenizing whole pieces of brain and separately characterizing the proteins, RNA, DNA, and other macromolecules within. While this has been sufficient to identify substantial changes, there is little ability to identify small changes or alterations that may occur in subsets of cells. To effectively investigate the biochemistry of disease in the brain, with its different cell types, we must first separate the cells and study them as phenotypically defined populations or even as individuals. In this project, we developed a new method for the generation of Whole Cell Dissociated Suspensions (WCDS) in fresh human brain tissue that could be shared as a resource with scientists to study single human cells or populations. Characterization of WCDS was done in paraffin-embedded sections stained with H&E, and by phenotyping with antibodies using immunohistochemistry and Fluorescence Activated Cell Sorting (FACS). Additionally, we compared extracted RNA from WCDS with RNA from adjacent intact cortical tissue, using RT-qPCR for cell-type-specific RNA for the same markers as well as whole transcriptome sequencing. More than 11,626 gene transcripts were successfully sequenced and classified using an external database either as being mainly expressed in neurons, astrocytes, microglia, oligodendrocytes, endothelial cells, or mixed (in two or more cell types). This demonstrates that we are currently capable of producing WCDS with a full representation of different brain cell types combined with RNA quality suitable for use in biochemical analysis.

Dynamic and cellular histomorphometry of trabeculae is the most biologically relevant way of assessing steady state bone health. Traditional measurement involves manual visual feature identification by a trained and qualified professional. Inherent with this methodology is the time and cost expenditure, as well as the subjectivity that naturally arises under human visual inspection. In this work, we propose a rapidly deployable, automated, and objective method for dynamic histomorphometry. We demonstrate that our method is highly effective in assessing cellular activities in distal femur and vertebra of mice which are injected with calcein and alizarin complexone 7 and 2 days prior to sacrifice. The mineralized bone tissues of mice are cryosectioned using a tape transfer protocol. A sequential workflow is implemented in which endogenous fluorescent signals (bone mineral, green and red mineralization lines), tartrate resistant acid phosphatase identified by ELF-97 and alkaline phosphatase identified by Fast Red are captured as individual tiled images of the section for each fluorescent color. All the images are then submitted to an image analysis pipeline that automates identification of the mineralized regions of bone and selection of a region of interest. The TRAP and AP stained images are aligned to the mineralized image using strategically placed fluorescent registration beads. Fluorescent signals are identified and are related to the trabecular surface within the ROI. Subsequently, the pipelined method computes static measurements, dynamic measurements, and cellular activities of osteoclast and osteoblast related to the trabecular surface. Our method has been applied to the distal femurs and vertebrae of 8 and 16 week old male and female C57Bl/6J mice. The histomorphometric results reveal a significantly greater bone turnover rate in female in contrast to male irrespective of age, validating similar outcomes reported by other studies.

Tissue engineering strategies, based on solid/porous scaffolds, suffer from several limitations, such as ineffective vascularization, poor cell distribution and organization within scaffold, in addition to low final cell density, among others. Therefore, the search for other tissue engineering approaches constitutes an active area of investigation. Decellularized matrices (DM) present major advantages compared to solid scaffolds, such as ideal chemical composition, the preservation of vascularization structure and perfect three-dimensional structure. In the present study, we aimed to characterize and investigate murine heart decellularized matrices as biomaterials for regular and whole organ tissue engineering. Heart decellularized matrices were characterized according to: 1. DNA content, through DNA quantificationo and PCR of isolated genomic DNA; 2. Histological structure, assessed after Hematoxylin and Eosin, as well as Masson's Trichrome stainings; 3. Surface nanostructure analysis, performed, using SEM. Those essays allowed us to conclude that DM was indeed decellularized, with preserved extracellular matrix structure. Following characterization, decellularized heart slices were seeded with induced Pluripotent Stem cells (iPS). As expected, but - to the best of our knowledge - never shown before, decellularization of murine heart matrices maintained matrix biocompatibility, as iPS cells rapidly attached to the surface of the material and proliferated. Strikingly though, heart DM presented a differentiation induction effect over those cells, which lost their pluripotency markers after 7 days of culture in the DM. Such loss of differentiation markers was observed, even though bFGF containing media mTSR was used during such period. Gene expression of iPS cells cultured on DM will be further analyzed, in order to assess the effects of culturing pluripotent stem cells in decellularized heart matrices.

Impaired elastic matrix remodeling occurs in reproductive tissues after vaginal delivery. This has been linked to development of pelvic organ prolapse (POP) for which there currently is no pharmacologic therapy. Hyaluronan oligomers and transforming growth factor beta 1 (termed elastogenic factors, EFs) have been shown to significantly enhance tropoelastin synthesis, elastic fiber assembly, and crosslinking by adult vascular smooth muscle cells (SMCs). The goal of this study was to ascertain if these factors similarly improve the quantity and quality of elastic matrix deposition by vaginal SMCs (VSMCs) isolated from lysyl oxidase like-1 knock out (LOXL1 KO) mouse model of POP. Cells isolated from whole vagina of a LOXL1 KO mouse (multiparous, stage 3 prolapse) were cultured and identified as SMCs by their expression of various SMC markers. Passage 2 vaginal SMCs (VSMCs; 3×10⁴/10 cm²) were cultured for 21 days with EFs. Cell layers and spent medium aliquots were assessed for elastin content and quality. EF-treated VSMCs proliferated at a similar rate to untreated controls but synthesized more total elastin primarily in the form of soluble matrix elastin. Elastin mRNA was also increased compared to controls. The elastic matrix was significantly denser in EF-treated cultures, which was composed of more mature, non-interrupted elastic fibers that were absent in controls. The results are promising towards development of a therapy to enhance regenerative elastic matrix repair in post-partum female pelvic floor tissues.

Therapeutic results of clinical autologous bone marrow cell (BMC) therapy trials for cardiac disease have been modest compared to results of BMC implantation into rodent hearts post-myocardial infarction (MI). In clinical trials, autologous BMCs are typically harvested from older patients who have recently suffered an MI. In contrast, experimental studies in rodent models typically utilize donor BMCs isolated from young, healthy, inbred mice that are not the recipients. Using unfractionated BMCs from donor mice at ages of young, middle-aged, and old, we discovered that recipient left ventricular function post-MI was significantly improved by young donor BMC implantation but was only preserved by middle-aged donor BMCs. Notably, old donor BMCs did not slow the decline in recipient post-MI cardiac function, suggesting BMC impairment by advanced donor age. Furthermore, we also show here that BMCs that are therapeutically impaired by donor age can be further impaired by concurrent donor MI. In conclusion, our findings suggest that therapeutic impairment of BMCs by advanced age is one of the important factors that can limit the success of clinical autologous BMC-based therapy.

BACKGROUND: Tissue engineered blood vessels could address the large clinical need for small caliber vascular grafts. Self-assembly approaches that employ transient scaffolds to form tissues from only cells and secreted matrix could form completely autologous vascular grafts that rapidly remodel and integrate with host tissue in vivo. The objective of this study was to develop a simple and rapid method to self-assemble vascular cells into vascular grafts. HYPOTHESIS: We hypothesized that entrapment in rapidly degrading fibrin gels could facilitate self-assembly of vascular smooth muscle cells into a tubular tissue comprised mainly of SMCs and secreted matrix. METHODS: Baboon SMCs were entrapped in fibrin around a silicone tube and cultured for 14 days without fibrinolysis inhibitor. Spontaneous delamination from the inner tube allowed for simple isolation of constructs with forceps. RESULTS: Engineered tissues are tubular, handleable, and highly cellular, with substantial collagen deposition. Fibrin is largely degraded within 14 days. Tensile elastic modulus of ring segments is 36.2 kPa and 1.60 MPa for the toe and heel regions of the stress-strain relation, respectively. CONCLUSION: Fibrin entrapment without fibrinolysis inhibitor can facilitate rapid self-assembly of SMCs into tubular tissues. Future work will focus on mechanical conditioning and co-culture with vascular endothelial cells to improve mechanical strength and impart antithrombogenicity.

Bone marrow derived mesenchymal stem cells (MSCs) have been shown to demonstrate benefit in multiple disease models characterized by inflammation such as sepsis and acute lung injury. Mechanistically we hypothesized that MSCs exhibit these properties through inhibition of leukocyte activation and modulation of leukocyte-endothelial interactions; key interlinked processes involved in the deleterious effects of injury and inflammation. In this paper we found that MSCs co-cultured with a monocytoid line, U937, inhibit U937 binding to pulmonary endothelial cells (PECs) stimulated with the inflammatory cytokine TNFα. Furthermore, we show that these effects on functional adhesion are not due to changes in inflammatory adhesion molecule expression on U937s. No changes were found in CD62L, CD29, CD11b and CD18 expression on U937s co-cultured with MSCs. To determine if the effects of MSCs on leukocyte-endothelial interactions are due to the effects of MSCs on leukocyte activation, we investigated whether MSCs affect functional activation of the transcription factor NF-Kappa B. We found that MSCs significantly inhibit transcriptional activation of NF-kappa B in U937s. We also found that MSCs inhibit DNA binding of NF-kappa B subunits p50 and p65 to putative NF-kappa B DNA binding sites. Concomitant with a decrease in NF-kappa B activation was a significant increase in IL-10, an anti-inflammatory cytokine known to inhibit activation of NF-kappa B. Taken together, these findings show that MSCs have potent effects on leukocyte-endothelial interactions which may be due to the direct effects of MSCs on IL-10 and NF-kB. These findings suggest a potential therapeutic role for MSCs in diseases characterized by inflammation such as acute lung injury or multi-organ failure induced by traumatic injury.