Co-reporter:Katelyn L. Sellgren
Journal of Biomedical Materials Research Part A 2015 Volume 103( Issue 8) pp:2509-2520
Publication Date(Web):
DOI:10.1002/jbm.a.35386
Abstract
Perfusion bioreactor plays important role in supporting 3D bone construct development. Scaffolds of chitosan composites have been studied to support bone tissue regeneration from osteogenic progenitor cells including human mesenchymal stem cells (hMSC). In this study, porous scaffolds of hydroxyapatite (H), chitosan (C), and gelatin (G) were fabricated by phase-separation and press-fitted in the perfusion bioreactor system where media flow is configured either parallel or transverse with respect to the scaffolds to investigate the impact of flow configuration on hMSC proliferation and osteogenic differentiation. The in vitro results showed that the interstitial flow in the transverse flow (TF) constructs reduced cell growth during the first week of culture but improved spatial cell distribution and early onset of osteogenic differentiation measured by alkaline phosphatase and expression of osteogenic genes. After 14 days of bioreactor culture, the TF constructs have comparable cell number but higher expression of bone markers genes and proteins compared to the parallel flow constructs. To evaluate ectopic bone formation, the HCG constructs seeded with hMSCs pre-cultured under two flow configurations for 7 days were implanted in CD-1 nude mice. While Masson's Trichrom staining revealed bone formation in both constructs, the TF constructs have improved spatial cell and osteoid distribution throughout the 2.0 mm constructs. The results highlight the divergent effects of media flow over the course of construct development and suggest that the flow configuration is an important parameter regulating the cellular events leading to bone construct formation in the HCG scaffolds. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 103A: 2509–2520, 2015.
Co-reporter:Teng Ma
Journal of Materials Chemistry A 2014 vol. 2(Issue 1) pp:31-35
Publication Date(Web):29 Oct 2013
DOI:10.1039/C3TB21369B
The functions of acellular biomaterials have evolved from a space filler and mechanical support to biological tissue replacements that guide endogenous tissue regeneration. Delivery of acellular materials avoids high cost of production and scientific and regulation barriers associated with cellular systems. The advent of tissue specific stem cells and advances in biomaterials have created new opportunities for refining the properties and identifying new applications for the acellular materials. In particular, increasing knowledge of mesenchymal stem cell in vivo identity and their roles in endogenous tissue regeneration has provided a target population of resident tissue stem cells and scientific foundation upon which the material properties can be optimized. This article discusses the progress in tissue-specific stem cells, their role in endogenous tissue repair, and methods that direct resident MSC migration and differentiation. These concepts are then discussed in the case of guided tissue regeneration to highlight the application and challenges of acellular biomaterials in clinical applications.
Co-reporter:Kimberly T. Hunter
Journal of Biomedical Materials Research Part A 2013 Volume 101A( Issue 4) pp:1016-1025
Publication Date(Web):
DOI:10.1002/jbm.a.34396
Abstract
Resorbable biomaterials have been investigated as barrier membranes to compartmentalize the periodontal defects while selectively guiding osteoprogenitor cell proliferation and bone tissue expansion. Hydroxyapatite (H), chitosan (C), and gelatin (G) have chemical similarity to the structural components of natural bone and their composites have been tested as bone scaffolds. Human mesenchymal stem or stromal cells (hMSCs) are inducible osteoprogenitors and are responsible for bone tissue repair and regeneration. In this study, the dynamic interactions of hMSC with composite hydroxyapatite–chitosan–gelatin (HCG) membranes were investigated. The association of HCG formed a biodegradable membrane with ∼60 wt % water and an initial stiffness of ∼20 kPa. Preconditioning in serum-containing media resulted in the formation nanopores in the HCG membranes and the increase of extracellular matrix (ECM) protein adsorption. Expression of integrin α2β1 and α5β1 coincided with ECM enrichment, suggesting the enhanced cell–ECM interactions. The elevated expression of bone marker proteins and genes in the HCG membranes suggests the progression of hMSC osteogenic differentiation in the absence of chemical induction. The results showed that the HCG membranes possess sufficient mechanical and structural properties to function as a barrier membrane, and that the adsorbed ECM proteins effectively functionalized the HCG membranes and promoted hMSC osteogenic differentiation. © 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.
Co-reporter:Junho Kim PhD
Journal of Cellular Biochemistry 2013 Volume 114( Issue 3) pp:716-727
Publication Date(Web):
DOI:10.1002/jcb.24413
Abstract
Human mesenchymal stromal or stem cells (hMSCs) are being investigated for cell therapy in a wide range of diseases. MSCs are a potent source of trophic factors and actively remodel their immediate microenvironment through the secretion of bioactive factors in response to external stimuli such as oxygen tension. In this study, we examined the hypothesis that hypoxia influences hMSC properties in part through the regulation of extracellular milieu characterized by the extracellular matrix (ECM) matrices and the associated fibroblast growth factor-2 (FGF-2). The decellularized ECM matrices derived from hMSC culture under both hypoxic (e.g., 2% O2) and the standard culture (e.g., 20% O2) conditions have different binding capacities to the cell-secreted and exogenenous FGF-2. The reduced hMSC proliferation in the presence of FGF-2 inhibitor and the differential capacity of the decellularized ECM matrices in regulating hMSC osteogeneic and adipogenic differentiation suggest an important role of the endogenous FGF-2 in sustaining hMSC proliferation and regulating hMSC fate. Additionally, the combination of the ECM adhesion and hypoxic culture preserved hMSC viability under serum withdrawal. Together, the results suggest the synergistic effect of hypoxia and the ECM matrices in sustaining hMSC ex vivo expansion and preserving their multi-potentiality and viability under nutrient depletion. The results have important implication in optimizing hMSC expansion and delivery strategies to obtain hMSCs in sufficient quantity with required potency and to enhance survival and function upon transplantation. J. Cell. Biochem. 114: 716–727, 2013. © 2012 Wiley Periodicals, Inc.
Co-reporter:Aiping Zhu, Feng Zhao, Teng Ma
Acta Biomaterialia 2009 Volume 5(Issue 6) pp:2033-2044
Publication Date(Web):July 2009
DOI:10.1016/j.actbio.2009.02.016
Abstract
Vascular graft surface properties significantly affect adhesion, growth and function of endothelial cells (ECs). The bulk degradation property of poly(lactic acid) (PLA) makes it possible for it to be replaced by cellular materials and PLA is desirable as a scaffold material for vascular grafts. However, PLA has an unfavorable surface property for EC adhesion and proliferation due to the lack of a selective cell adhesion motif. Photo-initiated surface-grafting polymerization is a promising method for immobilizing certain biomacromolecules on material surfaces without compromising bulk properties. N-Maleic acyl-chitosan (NMCS) is a novel biocompatible amphiphilic derivative of chitosan with double bonds and can be initiated by ultraviolet light. In this study, gelatin was complexed with NMCS via hydrophobic interaction, and gel/NMCS complex thus formed was then grafted on the PLA surface to improve EC biocompatibility. X-ray photoelectron and Fourier transform infrared spectroscopy, and water contact angle measurement confirmed immobilization of the gel/NMCS complex on PLA surface. Moreover, the gel/NMCS modified PLA enhanced human umbilical vein endothelial cell (HUVEC) spreading and flattening, and promoted the expression of more structured CD31 and vWF compared to unmodified PLA film. Compared to the unmodified PLA surface, the HUVECs on the modified PLA surface had elevated uptake of acetylated low-density lipoprotein, and maintained the ability to modulate metabolic activity upon exposure to shear stress at 5 dyn cm−2 by up-regulating nitric oxide and prostacyclin production. Cell retention was 1.6 times higher on the gel/NMCS–PLA surface, demonstrating its improved potential for hemocompatibility. These results indicate that photo-initiated surface-grafting of the biomimetic gel/NMCS complex is an effective method to modify material surfaces as vascular grafts.
Co-reporter:Feng Zhao, Warren L. Grayson, Teng Ma, Bruce Bunnell, William W. Lu
Biomaterials 2006 Volume 27(Issue 9) pp:1859-1867
Publication Date(Web):March 2006
DOI:10.1016/j.biomaterials.2005.09.031
Human mesenchymal stem cells (hMSCs) have great potential in bone tissue engineering, and hydroxyapatite (HA), a natural component of human hard tissues, is believed to support hMSC growth and osteogenic differentiation. In this study, two types of biomimetic composite materials, chitosan–gelatin (CG) and hydroxyapatite/chitosan–gelatin (HCG), were fabricated and compared to examine the effects of HA on hMSC adhesion and 3-D construct development. The 2-D membranes were prepared to examine the influence of HA on adhesion efficiency of hMSCs, while 3-D porous scaffolds were produced to investigate the effects of HA on material adsorption properties and 3-D hMSC construct development. HA was found to promote protein and calcium ion adsorption of the 3-D porous scaffolds in the complete tissue culture media. HMSCs exhibited higher initial cell adhesion efficiency to 2-D HCG membranes, and maintained higher proliferation rates in the 3-D porous HCG than CG scaffolds with 3.3 times higher final DNA amount in HCG scaffolds over a 35-day period. Colony forming unit-fibroblast (CFU-F) assays showed that higher percentages of cells maintained their progenicity in the 3-D porous HCG scaffolds over the 35-day culture period. Differentiation assays indicated that the multi-lineage differentiation potential of the hMSCs was preserved in both 3-D porous scaffolds. However, higher alkaline phosphate activity was detected in the 3-D porous HCG scaffolds upon osteogenic induction indicating improved osteogenic differentiation potential. The results demonstrate that enhanced protein and calcium ion adsorption properties of HA in the CG polymer network improve initial cell adhesion and long-term growth, favor osteogenic differentiation upon induction, as well as maintain the progenicity of the 3-D hMSC constructs.
Co-reporter:Yan Li, Teng Ma, Shang-Tian Yang, Douglas A Kniss
Biomaterials 2001 Volume 22(Issue 6) pp:609-618
Publication Date(Web):15 March 2001
DOI:10.1016/S0142-9612(00)00224-6
Nonwoven fibrous matrices have been widely used as scaffolds in tissue engineering, and modification of microstructure of these matrices is needed to organize cells in three-dimensional space with spatially balanced proliferation and differentiation required for functional tissue development. The method of thermal compression of nonwoven polyethylene terephthalate (PET) fabrics was developed and key parameters of temperature, pressure, and compression duration were evaluated in this study. The permanent deformation was obtained at elevated temperature under pressure and the viscoelastic compressional behaviors were observed, characterized by a distinct apparent modulus change in glass transition temperature region. A liquid extrusion method was further employed to analyze both pore size and its distribution for matrices with porosity ranging from 84 to 93%. It is also found that a more uniformly distributed pore size was resulted from thermal compression and the isotropic nature of nonwoven fabrics was preserved because of the proportional reduction of the pore by compression. The thermally compressed fabric matrices with two different pore sizes (15 and 20 μm in pore radius) were used to culture human trophoblast ED27 and NIH 3T3 cells. It was found that cells cultured in the different pore-size PET matrices had different cell spatial organization and proliferation rates. The smaller pores in the matrix allowed cells to spread better and proliferate faster, while cells in the larger pores tended to form large aggregates and had lower proliferation rate. The thermal compression technique also can be applied to other synthetic fibrous matrices including biodegradable polymers used in tissue engineering to modify the microstructure according to their viscoelastic properties.
Co-reporter:Jens T. Rosenberg, Katelyn L. Sellgren, Afi Sachi-Kocher, Fabian Calixto Bejarano, ... Samuel C. Grant
Cytotherapy (March 2013) Volume 15(Issue 3) pp:307-322
Publication Date(Web):1 March 2013
DOI:10.1016/j.jcyt.2012.10.013
Background aimsHuman mesenchymal stem cells (hMSCs) have gained interest for treatment of stroke injury. Using in vitro culture, the purpose of this study was to investigate the long-term detectability of hMSCs by magnetic resonance imaging (MRI) after transfection with a superparamagnetic iron oxide (SPIO) and evaluate the effects of SPIO on cellular activity, particularly under an ischemic environment.MethodshMSCs were exposed to low doses of SPIOs. After a short incubation period, cells were cultured for additional 1, 7 and 14 d to evaluate proliferation, colony formation and multilinear potential. Labeled cells were imaged and evaluated in agarose to quantify R2 and R2∗ contrast at each time point. Cells were placed in a low-oxygen, low-serum environment and tested for cytotoxicity. In addition, labeled cells were transplanted into an ischemic stroke model and evaluated with ex vivo MRI and histology.ResultsCellular events such as proliferation and differentiation were not affected at any of the exposures tested when cultured for 14 d. The low iron exposure and short incubation time are sufficient for detectability with MRI. However, the higher iron dosage results in higher calcification and cytotoxicity under in vitro ischemic conditions. Transplantation of the hMSCs labeled with an initial exposure of 22.4 μg of Fe showed excellent retention of contrast in stroke-induced rats.ConclusionsAlthough SPIO labeling is stable for long-term MRI detection and has limited effects on the multilineage potential of hMSCs, high-dose SPIO labeling may affect hMSC survival under serum and oxygen withdrawal.
Co-reporter:Jason J. Crowe, Samuel C. Grant, Timothy M. Logan, Teng Ma
Chemical Engineering Science (15 September 2011) Volume 66(Issue 18) pp:4138-4147
Publication Date(Web):15 September 2011
DOI:10.1016/j.ces.2011.05.046
Human mesenchymal stem cells (hMSCs) have significant potential for therapeutic tissue regeneration and repair. The creation of functional 3D constructs from hMSCs depends on the innate ability of MSCs to proliferate and differentiate, and is strongly influenced by the culture conditions. An inherent challenge in investigating 3D cellular construct development is the dynamic monitoring of the cellular and physiological environment over the course of construct formation. In this project, a novel 3D MR-compatible perfusion bioreactor using 3D poly(ethylene terephthalate) scaffolds was developed to provide such monitoring. The bioreactor system integrates cell seeding and growth, supports high density 3D tissue construct growth and facilitates repeated nuclear magnetic resonance (MR) signal acquisitions under both static and perfusion conditions. The reactor system also has the capacity to modulate macroscopic flow modes that simulates various tissue growth environments with repeated MR signal acquisition, providing the ability to gain insight into the dynamic interplay between the stem cells in the developing constructs and their microenvironment. Using 1H MR spectroscopy and MR imaging, localized spectroscopic data as well as imaging-based T2 and diffusion quantification were acquired from the hMSC growth construct for up to 40 days.Highlights► We developed a MR-compatible perfusion bioreactor to monitor stem cell construct development. ► The bioreactor integrates construct cultivation and MR monitoring in a single unit. ► The bioreactor can modulate macroscopic flow and allows repeated MR signal acquisitions. ► Parallel and transverse flow were introduced and monitored by MR.
Co-reporter:Teng Ma, Ang-Chen Tsai, Yijun Liu
Biochemical Engineering Journal (15 April 2016) Volume 108() pp:44-50
Publication Date(Web):15 April 2016
DOI:10.1016/j.bej.2015.07.014
•Large scale expansion is required in hMSC biomanufacture for clinical applications.•Culture-induced changes in hMSC property are a major barrier in hMSC translation.•Microenvironments significantly influence hMSC expansion and properties.•Understanding hMSC microenvironment is important in scaling up hMSC expansion.Human mesenchymal stem cells (hMSCs) are the primary candidate in cell therapy and have demonstrated significant potential in a wide range of diseases. The clinical translation of hMSC therapy requires robust and scalable expansion technology for the biomanufacturing of therapeutically competent cells. By nature, hMSCs are highly sensitive and responsive to the microenvironments and their therapeutic potency is significantly influenced by the culture conditions during expansion. Here, we discuss the emerging roles of microenvironments in regulating hMSC fate and the implication of regulating hMSC microenvironment to achieve scalable hMSC expansion in bioreactors.
Co-reporter:Ang-Chen Tsai, Yijun Liu, Teng Ma
Biochemical Engineering Journal (15 April 2016) Volume 108() pp:51-57
Publication Date(Web):15 April 2016
DOI:10.1016/j.bej.2015.09.002
•hMSCs were expanded in a 2.5 l CelliGen® 310 bioreactor for 9 days.•9.2-fold expansion was obtained with a 2.8-day population doubling time.•An exponential increase of glucose consumption and lactate production was measured.•Homogeneous cell distribution in the fibrous bed was observed.•hMSCs harvested from the bioreactor maintained their stem cell properties.Expansion of human mesenchymal stem cells (hMSCs) in bioreactor while preserving their innate properties is important in translation of hMSC-based therapy to clinical applications. The present study investigates the feasibility of hMSC expansion in a 2.5 L CelliGen® 310 Bioreactor packed with Fibra-Cel® disks. After 9 days of expansion, a 9.2-fold increase in cell number with the population doubling (PD) time of 2.8 days (67.2 h) was achieved and that the specific glucose consumption and lactate production were measured to be 12.48 pmol/cell/day and 20.95 pmol/cell/day, respectively. hMSCs harvested from the bioreactor maintained their properties based on the analysis of phenotypic surface markers, colony forming unit-fibroblasts (CFU-F) number, and multilineage differentiation ability. The results demonstrate the feasibility and the potential of the fibrous bed bioreactor for large scale hMSC expansion.
Co-reporter:Nathalie Muñoz, Junho Kim, Yijun Liu, Timothy M. Logan, Teng Ma
Journal of Biotechnology (10 January 2014) Volume 169() pp:95-102
Publication Date(Web):10 January 2014
DOI:10.1016/j.jbiotec.2013.11.010
•GC–MS was used to investigate glucose metabolism in hMSC and hMSC-derived osteoblasts (hMSC-OS) cultured at 2% or 20% O2.•hMSC and hMSC-OS exhibited different metabolic responses to low oxygen tension.•Hypoxia-induced inhibition of PDH was more pronounced in hMSC-OS compared to hMSC.•Hypoxia increased the apparent activity of the malate aspartate shuttle in hMSC, but not in hMSC-OS.Bone marrow derived human mesenchymal stem cells (hMSC) are the primary cell type in bone tissue engineering, and their life span during osteogenic differentiation is associated with changes in oxygen tension. As a ubiquitous regulator of cellular metabolic activity, oxygen tension influences the profiles of metabolites in the entire metabolic network and plays an important role in hMSC survival, function, and osteogenic differentiation. In the current study, we hypothesize that hMSC have a metabolic phenotype that supports growth in low oxygen environments and that this phenotype changes upon differentiation, leading to differential responses to oxygen tension. We developed a gas chromatography–mass spectrometry (GC–MS) based metabolic profiling approach to analyze the metabolic fate of 13C-glucose in glycolysis and the tricarboxylic acid cycle (TCA) in undifferentiated hMSC and hMSC-derived osteoblasts (hMSC-OS) in response to perturbation in oxygen tension; specifically we compared changes induced by culture under 20% vs. 2% O2. The isotope enrichments in the metabolites were calculated and used to infer activities of specific metabolic enzymes and the associated pathways. The results revealed contrasting metabolic profiles for hMSC and the hMSC-OS in both 20% and 2% O2 states, with the most significant differences involving coupling of glycolysis to the TCA cycle, glutaminolysis, and the malate-aspartate shuttle. The results have important implications in defining the optimal culture conditions for hMSC expansion and osteogenic differentiation.
Co-reporter:Teng Ma
Journal of Materials Chemistry A 2014 - vol. 2(Issue 1) pp:NaN35-35
Publication Date(Web):2013/10/29
DOI:10.1039/C3TB21369B
The functions of acellular biomaterials have evolved from a space filler and mechanical support to biological tissue replacements that guide endogenous tissue regeneration. Delivery of acellular materials avoids high cost of production and scientific and regulation barriers associated with cellular systems. The advent of tissue specific stem cells and advances in biomaterials have created new opportunities for refining the properties and identifying new applications for the acellular materials. In particular, increasing knowledge of mesenchymal stem cell in vivo identity and their roles in endogenous tissue regeneration has provided a target population of resident tissue stem cells and scientific foundation upon which the material properties can be optimized. This article discusses the progress in tissue-specific stem cells, their role in endogenous tissue repair, and methods that direct resident MSC migration and differentiation. These concepts are then discussed in the case of guided tissue regeneration to highlight the application and challenges of acellular biomaterials in clinical applications.
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