scholarly journals In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells

2020 ◽  
Vol 21 (2) ◽  
pp. 581 ◽  
Author(s):  
Phoonsuk Limraksasin ◽  
Takeru Kondo ◽  
Maolin Zhang ◽  
Hiroko Okawa ◽  
Thanaphum Osathanon ◽  
...  
Keyword(s):  
Cartilage Tissue ◽  
Marker Genes ◽  
Induction Medium ◽  
Mechanical Stimuli ◽  
Tissue Formation ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Chondrogenic Marker ◽  
Induced Pluripotent

Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol.

scholarly journals Optimizing Osteogenic Differentiation of Ovine Adipose-Derived Stem Cells by Osteogenic Induction Medium and FGFb, BMP2, or NELL1 In Vitro

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Emil Østergaard Nielsen ◽  
Li Chen ◽  
Jonas Overgaard Hansen ◽  
Matilda Degn ◽  
Søren Overgaard ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Stromal Cells ◽  
Basic Medium ◽  
Induction Medium ◽  
Control Analysis ◽  
Alizarin Red ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Fibroblast Growth Factor Basic

Although adipose-derived stromal cells (ADSCs) have been a major focus as an alternative to autologous bone graft in orthopedic surgery, bone formation potential of ADSCs is not well known and cytokines as osteogenic inducers on ADSCs are being investigated. This study aimed at isolating ADSCs from ovine adipose tissue (AT) and optimizing osteogenic differentiation of ovine ADSCs (oADSC) by culture medium and growth factors. Four AT samples were harvested from two female ovine (Texel/Gotland breed), and oADSCs were isolated and analyzed by flow cytometry for surface markers CD29, CD44, CD31, and CD45. Osteogenic differentiation was made in vitro by seeding oADSCs in osteogenic induction medium (OIM) containing fibroblast growth factor basic (FGFb), bone morphogenetic protein 2 (BMP2), or NEL-like molecule 1 (NELL1) in 4 different dosages (1, 10, 50, and 100 ng/ml, respectively). Basic medium (DMEM) was used as control. Analysis was made after 14 days by Alizarin red staining (ARS) and quantification. This study successfully harvested AT from ovine and verified isolated cells for minimal criteria for adipose stromal cells which suggests a feasible method for isolation of oADSCs. OIM showed significantly higher ARS to basic medium, and FGFb 10 ng/ml revealed significantly higher ARS to OIM alone after 14 days.

scholarly journals Mechanical Sensing Element PDLIM5 Promotes Osteogenesis of Human Fibroblasts by Affecting the Activity of Microfilaments

Biomolecules ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 759
Author(s):  
Xiaolan Huang ◽  
Rongmei Qu ◽  
Yan Peng ◽  
Yuchao Yang ◽  
Tingyu Fan ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Morphological Changes ◽  
Human Fibroblasts ◽  
Inhibitory Effect ◽  
Induction Medium ◽  
Sensing Element ◽  
Differentiation Potential ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction ◽  
Related Proteins

Human skin fibroblasts (HSFs) approximate the multidirectional differentiation potential of mesenchymal stem cells, so they are often used in differentiation, cell cultures, and injury repair. They are an important seed source in the field of bone tissue engineering. However, there are a few studies describing the mechanism of osteogenic differentiation of HSFs. Here, osteogenic induction medium was used to induce fibroblasts to differentiate into osteoblasts, and the role of the mechanical sensitive element PDLIM5 in microfilament-mediated osteogenic differentiation of human fibroblasts was evaluated. The depolymerization of microfilaments inhibited the expression of osteogenesis-related proteins and alkaline phosphatase activity of HSFs, while the polymerization of microfilaments enhanced the osteogenic differentiation of HSFs. The evaluation of potential protein molecules affecting changes in microfilaments showed that during the osteogenic differentiation of HSFs, the expression of PDLIM5 increased with increasing induction time, and decreased under the state of microfilament depolymerization. Lentivirus-mediated PDLIM5 knockdown by shRNA weakened the osteogenic differentiation ability of HSFs and inhibited the expression and morphological changes of microfilament protein. The inhibitory effect of knocking down PDLIM5 on HSF osteogenic differentiation was reversed by a microfilament stabilizer. Taken together, these data suggest that PDLIM5 can mediate the osteogenic differentiation of fibroblasts by affecting the formation and polymerization of microfilaments.

scholarly journals Decellularized Human Stromal Lenticules Combine with Corneal Epithelial-Like Cells: A New Resource for Corneal Tissue Engineering

2019 ◽  
Vol 2019 ◽  
pp. 1-10 ◽  
Author(s):  
Shuai Qin ◽  
Shuai Zheng ◽  
Bing Qi ◽  
Rui Guo ◽  
Guanghui Hou
Keyword(s):  
Tissue Engineering ◽  
Induction Medium ◽  
Nuclear Materials ◽  
Corneal Tissue ◽  
Corneal Epithelial ◽  
Human Ipscs ◽  
Immunological Rejection ◽  
Lenticule Extraction ◽  
Induced Pluripotent

The lack of donor corneal tissue or the immunological rejection remains a challenge for individuals with limbal stem cell deficiency (LSCD) who are treated with keratoplasty. Numerous lenticules which were extracted by small incision lenticule extraction (SMILE) appear to be useful materials for keratoplasty. In order to reduce the incidence of allograft rejection, lenticules would be decellularized. Lenticules which were treated with liquid nitrogen and nucleases had no cellular and nuclear materials remained. Human induced pluripotent stem cells (iPSCs) can be generated from the patient who requires keratoplasty, offering an autologous alternative and eliminating the risk of graft rejection. We found that BMP-4, RA, N-2 supplement, hEGF, B27, decellularized human stromal lenticules, conditioned medium, or induction medium promoted the differentiation of human iPSCs with high purity. The results showed that human iPSCs cultured for 4 days in differentiation medium A, 14 days in condition medium, and 1 week in induction medium on decellularized human stromal lenticules developed markedly higher expression of the markers P63, CK3, and CK12 than did those in the other methods. The level of gene expression of the epithelial and pluripotency markers and analysis by scanning electron microscopy and immunohistochemistry also showed successful differentiation. After inducing differentiation in vitro, corneal epithelial-like cells were induced. In the study, we investigated the possibility of a new resource for corneal tissue engineering.

scholarly journals Dual Delivery of TGF-β3 and Ghrelin in Microsphere/Hydrogel Systems for Cartilage Regeneration

Molecules ◽  
2021 ◽  
Vol 26 (19) ◽  
pp. 5732
Author(s):  
Jianjing Lin ◽  
Li Wang ◽  
Jianhao Lin ◽  
Qiang Liu
Keyword(s):  
Growth Factors ◽  
Chondrogenic Differentiation ◽  
Transforming Growth Factor ◽  
Double Emulsion ◽  
Cartilage Regeneration ◽  
Cartilage Tissue ◽  
Self Healing ◽  
Tissue Formation ◽  
Extraction Technology

Articular cartilage (AC) damage is quite common, but due to AC’s poor self-healing ability, the damage can easily develop into osteoarthritis (OA). To solve this problem, we developed a microsphere/hydrogel system that provides two growth factors that promote cartilage repair: transforming growth factor-β3 (TGF-β3) to enhance cartilage tissue formation and ghrelin synergy TGF-β to significantly enhance the chondrogenic differentiation. The hydrogel and microspheres were characterized in vitro, and the biocompatibility of the system was verified. Double emulsion solvent extraction technology (w/o/w) is used to encapsulate TGF-β3 and ghrelin into microspheres, and these microspheres are encapsulated in a hydrogel to continuously release TGF-β3 and ghrelin. According to the chondrogenic differentiation ability of mesenchymal stem cells (MSCs) in vitro, the concentrations of the two growth factors were optimized to promote cartilage regeneration.

scholarly journals Advancing cardiovascular tissue engineering

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 1045 ◽  
Author(s):  
George A. Truskey
Keyword(s):  
Tissue Engineering ◽  
Cardiac Tissue ◽  
Great Promise ◽  
Mechanical Stimuli ◽  
Cardiovascular Tissue ◽  
Microphysiological Systems ◽  
Cardiovascular Tissue Engineering ◽  
Induced Pluripotent ◽  
Improved Methods

Cardiovascular tissue engineering offers the promise of biologically based repair of injured and damaged blood vessels, valves, and cardiac tissue. Major advances in cardiovascular tissue engineering over the past few years involve improved methods to promote the establishment and differentiation of induced pluripotent stem cells (iPSCs), scaffolds from decellularized tissue that may produce more highly differentiated tissues and advance clinical translation, improved methods to promote vascularization, and novel in vitro microphysiological systems to model normal and diseased tissue function. iPSC technology holds great promise, but robust methods are needed to further promote differentiation. Differentiation can be further enhanced with chemical, electrical, or mechanical stimuli.

Impaired In Vitro Osteogenesis in Multiple Myeloma Bone Marrow Osteoprogenitor Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2349-2349
Author(s):  
Nandita Bose ◽  
Rachel A. Kahler ◽  
Xiaodong Li ◽  
Hope Bergemann ◽  
Vincent Rajkumar ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Dna Binding ◽  
Splice Variant ◽  
Immunoblot Analysis ◽  
Induction Medium ◽  
Rt Pcr ◽  
Runx2 Expression ◽  
Osteogenic Induction Medium ◽  
Osteogenic Induction

Abstract In individuals with the plasma cell malignancy multiple myeloma, there is evidence that bone formation rates are reduced and that increased ostoeclastic bone resorption is associated with impaired osteoblast function. We investigated the osteogenic differentiation capacity of myeloma bone marrow mesenchymal progenitor cells (MPCs). We also examined the expression levels and activity of Runx2, the transcription factor required for osteogenesis. Bone marrow MPCs were cultured in osteogenic induction medium and assessed for bone alkaline phosphatase (bALP) expression (Sigma kit), induction of osteoblast specific genes like osteocalcin (RT-PCR) and mineralization by von Kossa staining. Immunoblot analysis and electrophoretic mobility shift assays (EMSA) were used to determine Runx2 expression and DNA-binding activity respectively. RT-PCR was used to sequence Runx2 and detect any mutations or deletions present within its domains. Transactivation ability of Runx2 was measured by its ability to activate osteocalcin promoter in transient transfection assays. Myeloma -derived MPCs showed reduced levels of bALP and osteocalcin transcript and a lower degree of mineralization, in osteogenic induction medium, as compared to that of the healthy donors. Immunoblot analysis and EMSA indicated equivalent Runx2 expression and DNA-binding capacity, respectively, in both healthy donor and myeloma-derived MPCs. Sequence analysis of Runx2 indentified a splice variant of Runx2 lacking exon 8 (Runx2D8) in myeloma patients with reduced transactivation ability. Co-transfection of the splice variant led to reduced transcriptional activity of the full-length Runx2. The reduced transactivation ability of spliced Runx2, as well as its suppressive action on the transactivation function of full-length Runx2, likely contributes to the defective osteogenesis clinically observed in myeloma pateints.

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