Traumatized muscle‐derived multipotent progenitor cells recruit endothelial cells through vascular endothelial growth factor‐A action

Traumatized muscle, such as that debrided from blast injury sites, is considered a promising and convenient tissue source for multipotent progenitor cells (MPCs), a population of adult mesenchymal stem cell (MSC)‐like cells. The present study aimed to assess the regenerative therapeutic potential of human traumatized muscle‐derived MPCs, e.g., for injury repair in the blast‐traumatized extremity, by comparing their pro‐angiogenic potential in vitro and capillary recruitment activity in vivo to those of MSCs isolated from human bone marrow, a widely‐used tissue source. MPCs were tested for their direct and indirect effects on human microvascular endothelial cells (ECs) in vitro. The findings reported here showed that MPC‐conditioned culture medium (MPC‐CM), like MSC‐CM, promoted EC‐cord network branching. Silent (si)RNA‐mediated silencing of vascular endothelial growth factor‐A (VEGF‐A) expression in MPCs attenuated this effect. In a chick embryonic chorioallantoic membrane in vivo angiogenesis assay, MPCs encapsulated in photocrosslinked gelatin scaffold recruited blood vessels more efficiently than either MSCs or human foreskin fibroblasts. Together, these findings support the potential application of traumatized muscle‐derived MPCs in cell‐based regenerative medicine therapies as a result of their influence on EC organization. Copyright © 2017 John Wiley & Sons, Ltd.     

Agonism of Wnt–β‐catenin signalling promotes mesenchymal stem cell (MSC) expansion

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell‐based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6‐bromoindirubin‐3′‐oxime (BIO) increases MSC β‐catenin activity 106‐fold and stem cell‐associated gene expression ~33‐fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8‐fold in typical 2D culture conditions, as well as 1.3‐fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIO's potential utility as a proliferative agent for cell transplantation and tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.     

Epidermal‐like architecture obtained from equine keratinocytes in three‐dimensional cultures

Despite the high prevalence of skin conditions in the horse, there is a dearth of literature on the culture and biology of equine skin cells, and this is partially attributable to the lack of suitable in vitro skin models. The objective of this study was to develop a three‐dimensional (3D) culture system that would support the proliferation and differentiation of equine keratinocytes, similar to that observed in natural epidermis. Cell monolayers were obtained from explants of equine skin and serially passaged as highly pure keratinocyte populations (> 95% of cells), based on their expression of cytokeratins, including CK‐5 and CK‐14, which are associated in vivo with proliferating keratinocyte populations. Explant‐derived keratinocytes were seeded into Alvetex? 3D tissue scaffolds for 30 days under conditions that promote cell differentiation. Ultrastructural, immunohistochemical and biochemical analyses revealed that keratinocytes within scaffolds were able to proliferate and attain tissue polarity, including differentiation into basal and suprabasal layers. The basal layer contained distinct cuboidal cells with large nuclei and stained for proliferative markers such as CK‐5 and CK‐14. In contrast, the suprabasal layers consisted of cells with distinct polyhedral morphology, abundant cytoplasmic processes and desmosomes indicative of stratum spinosum and distinct flattened cornified cells that expressed involucrin, a marker of terminal differentiation. Thus, keratinocytes derived from primary equine skin explants were able to attain epidermal‐like architecture in culture. This novel system could provide a very useful tool for modelling skin diseases, drug testing/toxicity studies and, potentially, equine regenerative medicine. Copyright © 2016 John Wiley & Sons, Ltd.     

Serum‐free bioprocessing of adult human and rodent skin‐derived Schwann cells: implications for cell therapy in nervous system injury

Peripheral nerve injury affects 2.8% of trauma patients with severe cases often resulting in long‐lived permanent disability, despite nerve repair surgery. Autologous Schwann cell (SC) therapy currently provides an exciting avenue for improved outcomes for these patients, particularly with the possibility to derive SCs from easily‐accessible adult skin. However, due to current challenges regarding the efficient expansion of these cells, further optimization is required before they can be seriously considered for clinical application. Here, a microcarrier‐based bioreactor system is proposed as a means to scale‐up large numbers of adult skin‐derived SCs for transplantation into the injured nerve. Bioprocessing parameters that allow for the expansion of adult rodent SCs have been identified, whilst maintaining similar rates of proliferation (as compared to static‐grown SCs), expression of SC markers, and, importantly, their capacity to myelinate axons following transplant into the injured sciatic nerve. The same bioprocessing parameters can be applied to SCs derived from adult human skin, and like rodent cells, they sustain their proliferative potential and expression of SC markers. Taken together, this dataset demonstrates the basis for a scalable bioprocess for the production of SCs, an important step towards clinical use of these cells as an adjunct therapy for nerve repair. Copyright © 2017 John Wiley & Sons, Ltd.     

One‐day treatment of small molecule 8‐bromo‐cyclic AMP analogue induces cell‐based VEGF production for in vitro angiogenesis and osteoblastic differentiation

Small molecule‐based regenerative engineering is emerging as a promising strategy for regenerating bone tissue. Small molecule cAMP analogues have been proposed as novel biofactors for bone repair and regeneration and, while promising, the effect that these small molecules have on angiogenesis, a critical requirement for successful bone regeneration, is still unclear. Our previous research demonstrated that the small molecule cAMP analogue 8‐bromoadenosine‐3′,5′‐cyclic monophosphate (8‐Br‐cAMP) was able to promote initial osteoblast adhesion on a polymeric scaffold via cAMP signalling cascades. Here, we report that 8‐Br‐cAMP is capable of inducing in vitro cell‐based VEGF production for angiogenesis promotion. We first demonstrated that treating osteoblast‐like MC3T3‐E1 cells with 8‐Br‐cAMP for 1 day significantly increased VEGF production and secretion. We then demonstrated that 8‐Br‐cAMP‐induced cell‐secreted VEGF is biologically active and may promote angiogenesis, as evidenced by increased human umbilical vein endothelial cells (HUVECs) migration and tubule formation. In addition, treatment of MC3T3‐E1 cells with 8‐Br‐cAMP for as short as a single day resulted in enhanced ALP activity as well as matrix mineralization, demonstrating in vitro osteoblastic differentiation. A short‐term 8‐Br‐cAMP treatment also addresses the concern of non‐specific cytotoxicity, as our data indicate that a 1‐day 8‐Br‐cAMP treatment scheme supports cellular proliferation of MC3T3‐E1 cells as well as HUVECs. While the major concern associated with small molecule drugs is the risk of non‐specific cytotoxicity, the short exposure treatment outlined in this paper provides a very promising strategy to mitigate the risk associated with small molecules. Copyright © 2013 John Wiley & Sons, Ltd.     

Interactive endothelial phenotype maintenance and osteogenic differentiation of adipose tissue stromal vascular fraction SSEA‐4+‐derived cells

Bone formation relies on complex processes that require well‐orchestrated interactions between several cell types, such as bone‐forming cells (osteoblasts, OBs) and endothelial cells (ECs). Their co‐culture has been proved relevant to mimicking specific features of the bone niche. Here we propose the co‐culture of microvascular‐like ECs and pre‐OBs, both derived from the SSEA‐4⁺ cell subpopulation from the stromal vascular fraction of human adipose tissue (SSEA‐4⁺ hASCs), to define the conditions in which cells synergistically communicate to support the full differentiation of pre‐OBs and maintenance of the EC phenotype. Co‐cultures of different ratios of the two cell types were established and maintained for up to 21 days in standard endothelial maintenance (ENDO) and osteogenic differentiation (OST) media, as well as in a mixture of these (MIX). The osteogenic maturation of pre‐OBs (ALP activity, OPN and OCN expression, calcium deposition), the evolution of EC numbers (CD31⁺ cells) and maintenance of the endothelial phenotype (CD31 and vWF expression, LDL uptake) were assessed throughout the culture time as a function of cell ratio and culture media. The results obtained demonstrate that EC number has a significant effect on the osteogenic differentiation of pre‐OBs, depending on the medium used. While in ENDO medium the osteogenic differentiation was not observed, in the OST and MIX media it was attained at similar levels, except for the co‐culture with a higher number of ECs in MIX medium. These findings demonstrate that the use of SSEA‐4⁺ hASCs as a single‐cell source is promising to attain 3D bone‐like models with the potential to promote vascularized bone tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Cryopreserved ECP‐treated lymphocytes maintain apoptotic response and anti‐proliferative effect

Background : The ability to cryopreserve a portion of the cells treated during extracorporeal photopheresis (ECP) would improve therapy logistics, particularly for pediatric patients, by allowing multiple therapeutic doses to be collected from a single apheresis session. However, the effect of cryopreservation on ECP‐treated cells is unknown (e.g., ECP‐induced lymphocyte apoptosis and inhibition of proliferation). Study Design and Methods : Mononuclear cell (MNC) apheresis products collected from healthy subjects were ECP‐treated using offline methods. Fresh samples of ECP‐treated and control cells were placed immediately in culture. The remainder of the cells were frozen in cryovials (n = 8) or cryobags (n = 8) at ?80°C. After 1 week of ?80°C storage, ECP‐treated and control cells were thawed rapidly and samples were placed in culture. Lymphocyte apoptosis was assessed by phosphatidylserine exposure using Annexin V/7‐AAD labeling. Lymphocyte proliferation after 3 days culture was measured using the carboxyfluorescein succinimidyl ester labeling technique. Results : On Day 0, apoptosis levels were <5% in fresh ECP‐treated and control cells and approximately 20% on thawing of cryopreserved ECP‐treated and control cells. Apoptosis levels were comparable between the two cryopreserved groups immediately on thawing, indicating that ECP‐treated cells were no more sensitive to the cryopreservation process than control cells. During 72‐h culture, apoptosis levels increased to >80% in fresh and cryopreserved ECP‐treated cells but remained near constant in both control groups. Inhibition of lymphocyte proliferation was >95% in all ECP‐treated cells with no significant difference between fresh and cryopreserved cells (P = 0.12). Conclusion : Cryopreservation did not impair the apoptotic response or anti‐proliferative effect of ECP‐treated lymphocytes, thereby demonstrating early feasibility of this approach. J. Clin. Apheresis 30:154–161, 2015. © 2014 Wiley Periodicals, Inc.     

Evaluation of in vitro spermatogenesis using poly(D,L‐lactic‐co‐glycolic acid) (PLGA)‐based macroporous biodegradable scaffolds

Successful in vitro differentiation of spermatogenic cells into spermatids appears to offer extremely attractive potential for the treatment of impaired spermatogenesis and male infertility. Experimental evidence indicates that biocompatible polymers may improve in vitro reconstitution and regeneration of tissues of various origins. Here, we fabricated highly porous biodegradable poly(D,L ‐lactic‐co‐glycolic acid) or PLGA co‐polymer scaffolds by combining the gas‐foaming and salt‐leaching methods, using ammonium bicarbonate as a porogen, which allowed us to generate polymer scaffolds with a high density of interconnected pores of 400–500 µm in average diameter, concomitant with a high malleability to mould a wide range of temporal tissue scaffolds requiring a specific shape and geometry. The PLGA scaffolds were biocompatible and biodegradable, as evidenced by the fact that they survived almost 3 month long subcutaneous xenografting into immunodeficient host mice and became easily destroyable after recovery. Immature rat testicular cells that were seeded onto the surface of the scaffold exhibited about 65% seeding efficiency and up to 75% viability after 18 days in culture. Furthermore, our scaffolds enhanced the proliferation and differentiation of spermatogenic germ cells to a greater extent than conventional in vitro culture methods, such as monolayer or organ culture. Taken together, an implication of the present findings is that the PLGA‐based macroporous scaffold may provide a novel means by which spermatocytes could be induced to differentiate into presumptive spermatids. Copyright © 2010 John Wiley & Sons, Ltd.     

Ameloblasts serum‐free conditioned medium: bone morphogenic protein 4‐induced odontogenic differentiation of mouse induced pluripotent stem cells

Induced pluripotent stem (iPS) cells possess the ability of self‐renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum‐free conditioned medium (ASF–CM), we successfully generated ameloblast‐like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF–CM supplemented with BMP4 (ASF–BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast‐specific as well as odontoblast‐specific genes. On the other hand, culturing mouse iPS cells in ASF–CM supplemented with noggin (ASF–noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF–BMP4 to differentiate into ameloblast‐like and odontoblast‐like cells. The results of our study raise the possibility of using patient‐specific iPS cells for tooth regeneration in the future. Copyright © 2016 John Wiley & Sons, Ltd.     

Biocompatibility of two model elastin‐like recombinamer‐based hydrogels formed through physical or chemical cross‐linking for various applications in tissue engineering and regenerative medicine

Biocompatibility studies, especially innate immunity induction, in vitro and in vivo cytotoxicity, and fibrosis, are often lacking for many novel biomaterials including recombinant protein‐based ones, such as elastin‐like recombinamers (ELRs), and has not been extensively explored in the scientific literature, in contrast to traditional biomaterials. Herein, we present the results from a set of experiments designed to elucidate the preliminary biocompatibility of 2 types of ELRs that are able to form extracellular matrix‐like hydrogels through either physical or chemical cross‐linking both of which are intended for different applications in tissue engineering and regenerative medicine. Initially, we present in vitro cytocompatibility results obtained upon culturing human umbilical vein endothelial cells on ELR substrates, showing optimal proliferation up to 9 days. Regarding in vivo cytocompatibility, luciferase‐expressing hMSCs were viable for at least 4 weeks in terms of bioluminescence emission when embedded in ELR hydrogels and injected subcutaneously into immunosuppressed mice. Furthermore, both types of ELR‐based hydrogels were injected subcutaneously in immunocompetent mice and serum TNFα, IL‐1β, IL‐4, IL‐6, and IL‐10 concentrations were measured by enzyme‐linked immunosorbent assay, confirming the lack of inflammatory response, as also observed upon macroscopic and histological evaluation. All these findings suggest that both types of ELRs possess broad biocompatibility, thus making them very promising for tissue engineering and regenerative medicine‐related applications.     

Traumatic sternal segment dislocation in a 3‐year‐old girl: Sonographic findings

Sternal fractures are uncommon in the pediatric population, and sternal segment dislocations are even rarer with only a few cases reported in the literature. Most cases are secondary to direct trauma to the chest, but nontraumatic dislocations have been reported. The diagnosis can be difficult to establish with standard radiographs, while CT is not desirable in the pediatric population due to the associated irradiation. Ultrasound (US) can be used as the first‐line modality to evaluate the sternum. We report the US findings associated with a case of traumatic sternal segment dislocation in a 3‐year‐old girl. © 2016 Wiley Periodicals, Inc. J Clin Ultrasound 45 :45–49, 2017     

SIRT1‐dependent anti‐senescence effects of cell‐deposited matrix on human umbilical cord mesenchymal stem cells

Human umbilical cord‐derived mesenchymal stem cells (UC‐MSCs) are considered an attractive cell source for tissue regeneration. However, environmental oxidative stress can trigger premature senescence in MSCs and thus compromises their regenerative potential. Extracellular matrix (ECM) derived from MSCs has been shown to facilitate cell proliferation and multi‐lineage differentiation. This investigation evaluated the effect of cell‐deposited decellularized ECM (DECM) on oxidative stress‐induced premature senescence in UC‐MSCs. Sublethal dosages of H₂O₂, ranging from 50 μm to 200 μm , were used to induce senescence in MSCs. We found that DECM protected UC‐MSCs from oxidative stress‐induced premature senescence. When treated with H₂O₂ at the same concentration, cell proliferation of DECM‐cultured UC‐MSCs was twofold higher than those on standard tissue culture polystyrene (TCPS). After exposure to 100 μm H₂O₂, fewer senescence‐associated β‐galactosidase‐positive cells were observed on DECM than those on TCPS (17.6  ±  4.0% vs. 60.4  ±  6.2%). UC‐MSCs cultured on DECM also showed significantly lower levels of senescence‐related regulators, such as p16^INK4α and p21. Most importantly, DECM preserved the osteogenic differentiation potential of UC‐MSCs with premature senescence. The underlying molecular mechanisms involved the silent information regulator type 1 (SIRT1)‐dependent signalling pathway, confirmed by the fact that the SIRT1 inhibitor nicotinamide counteracted the DECM‐mediated anti‐senescent effect. Collagen type I, rather than fibronectin, partially contributed to the protective effect of decellularized matrix. These findings provide a new strategy of using stem cell‐deposited matrix to overcome the challenge of cellular senescence and to facilitate the clinical application of MSCs in regenerative medicine. Copyright © 2017 John Wiley & Sons, Ltd.     

2‐(Trimethylammonium)ethyl (R)‐3‐methoxy‐3‐oxo‐2‐stearamidopropyl phosphate promotes megakaryocytic differentiation of myeloid leukaemia cells and primary human CD34+ haematopoietic stem cells

In this study we showed that 2‐(t rimethylammonium)e thyl (R)‐3‐m ethoxy‐3‐o xo‐2‐s tearamidopropyl pho sphate [(R)‐TEMOSPho], a derivative of an organic chemical identified from a natural product library, promotes highly efficient differentiation of megakaryocytes. Specifically, (R)‐TEMOSPho induces cell cycle arrest, cell size increase and polyploidization from K562 and HEL cells, which are used extensively to model megakaryocytic differentiation. In addition, megakaryocyte‐specific cell surface markers showed a dramatic increase in expression in response to (R)‐TEMOSPho treatment. Importantly, we demonstrated that such megakaryocytic differentiation can also be induced from primary human CD34⁺ haematopoietic stem cells. Activation of the PI3K–AKT pathway and, to a lesser extent, the MEK–ERK pathway appears to be required for this process, as blocking with specific inhibitors interferes with the differentiation of K562 cells. A subset of (R)‐TEMOSPho‐treated K562 cells undergoes spontaneous apoptosis and produces platelets that are apparently functional, as they bind to fibrinogen, express P‐selectin and aggregate in response to SFLLRN and AYPGFK, the activating peptides for the PAR1 and PAR4 receptors, respectively. Taken together, these results indicate that (R)‐TEMOSPho will be useful for dissecting the molecular mechanisms of megakaryocytic differentiation, and that this class of compounds represents potential therapeutic reagents for thrombocytopenia. Copyright © 2012 John Wiley & Sons, Ltd.     

Chondrogenic potential of injectable κ‐carrageenan hydrogel with encapsulated adipose stem cells for cartilage tissue‐engineering applications

Due to the limited self‐repair capacity of cartilage, regenerative medicine therapies for the treatment of cartilage defects must use a significant amount of cells, preferably applied using a hydrogel system that can promise their delivery and functionality at the specific site. This paper discusses the potential use of κ‐carrageenan hydrogels for the delivery of stem cells obtained from adipose tissue in the treatment of cartilage tissue defects. The developed hydrogels were produced by an ionotropic gelation method and human adipose stem cells (hASCs) were encapsulated in 1.5% w/v κ‐carrageenan solution at a cell density of 5 × 10⁶ cells/ml. The results from the analysis of the cell‐encapsulating hydrogels, cultured for up to 21 days, indicated that κ‐carrageenan hydrogels support the viability, proliferation and chondrogenic differentiation of hASCs. Additionally, the mechanical analysis demonstrated an increase in stiffness and viscoelastic properties of κ‐carrageenan gels with their encapsulated cells with increasing time in culture with chondrogenic medium. These results allowed the conclusion that κ‐carrageenan exhibits properties that enable the in vitro functionality of encapsulated hASCs and thus may provide the basis for new successful approaches for the treatment of cartilage defects. Copyright © 2013 John Wiley & Sons, Ltd.     

In vitro biocompatibility of 45S5 Bioglass®‐derived glass–ceramic scaffolds coated with poly(3‐hydroxybutyrate)

The aim of this work was to study the in vitro biocompatibility of glass–ceramic scaffolds based on 45S5 Bioglass^®, using a human osteosarcoma cell line (HOS‐TE85). The highly porous scaffolds were produced by the foam replication technique. Two different types of scaffolds with different porosities were analysed. They were coated with a biodegradable polymer, poly(3‐hydroxybutyrate) (P(3HB)). The scaffold bioactivity was evaluated by soaking in a simulated body fluid (SBF) for different durations. Compression strength tests were performed before and after immersion in SBF. These experiments showed that the scaffolds are highly bioactive, as after a few days of immersion in SBF a hydroxyapatite‐like layer was formed on the scaffold's surface. It was also observed that P(3HB)‐coated samples exhibited higher values of compression strength than uncoated samples. Biocompatibility assessment was carried out by qualitative evaluation of cell morphology after different culture periods, using scanning electron microscopy, while cell proliferation was determined by using the AlamarBlue^? assay. Alkaline phosphatase (ALP) and osteocalcin (OC) assays were used as quantitative in vitro indicators of osteoblast function. Two different types of medium were used for ALP and OC tests: normal supplemented medium and osteogenic medium. HOS cells were seeded and cultured onto the scaffolds for up to 2 weeks. The AlamarBlue assay showed that cells were able to proliferate and grow on the scaffold surface. After 7 days in culture, the P(3HB)‐coated samples had a higher number of cells on their surfaces than the uncoated samples. Regarding ALP‐ and OC‐specific activity, no significant differences were found between samples with different pore sizes. All scaffolds containing osteogenic medium seemed to have a slightly higher level of ALP and OC concentration. These experiments confirmed that Bioglass^®/P(3HB) scaffolds have potential as osteoconductive tissue engineering substrates for maintenance and normal functioning of bone tissue. Copyright © 2009 John Wiley & Sons, Ltd.     

Culture effects of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) on cryopreserved human adipose‐derived stromal/stem cell proliferation and adipogenesis

Previous studies have demonstrated that EGF and bFGF maintain the stem cell properties of proliferating human adipose‐derived stromal/stem cells (hASCs) in vitro. While the expansion and cryogenic preservation of isolated hASCs are routine, these manipulations can impact their proliferative and differentiation potential. This study examined cryogenically preserved hASCs (n = 4 donors), with respect to these functions, after culture with basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) at varying concentrations (0–10 ng/ml). Relative to the control, cells supplemented with EGF and bFGF significantly increased proliferation by up to three‐fold over 7–8 days. Furthermore, cryopreserved hASCs expanded in the presence of EGF and bFGF displayed increased oil red O staining following adipogenic induction. This was accompanied by significantly increased levels of several adipogenesis‐related mRNAs: aP2, C/EBPα, lipoprotein lipase (LPL), PPARγ and PPARγ co‐activator‐1 (PGC1). Adipocytes derived from EGF‐ and bFGF‐cultured hASCs exhibited more robust functionality based on insulin‐stimulated glucose uptake and atrial natriuretic peptide (ANP)‐stimulated lipolysis. These findings indicate that bFGF and EGF can be used as culture supplements to optimize the proliferative capacity of cryopreserved human ASCs and their adipogenic differentiation potential. Copyright © 2009 John Wiley & Sons, Ltd.     

Additive manufacturing of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] scaffolds for engineered bone development

A wide range of poly(hydroxyalkanoate)s (PHAs), a class of biodegradable polyesters produced by various bacteria grown under unbalanced conditions, have been proposed for the fabrication of tissue‐engineering scaffolds. In this study, the manufacture of poly[(R)‐3‐hydroxybutyrate‐co‐(R)‐3‐hydroxyhexanoate] (or PHBHHx) scaffolds, by means of an additive manufacturing technique based on a computer‐controlled wet‐spinning system, was investigated. By optimizing the processing parameters, three‐dimensional scaffolds with different internal architectures were fabricated, based on a layer‐by‐layer approach. The resulting scaffolds were characterized by scanning electron microscopy, which showed good control over the fibre alignment and a fully interconnected porous network, with porosity in the range 79–88%, fibre diameter 47–76 µm and pore size 123–789 µm. Moreover, the resulting fibres presented an internal porosity connected to the external fibre surface as a consequence of the phase‐inversion process governing the solidification of the polymer solution. Scaffold compressive modulus and yield stress and strain could be varied in a certain range by changing the architectural parameters. Cell‐culture experiments employing the MC3T3‐E1 murine pre‐osteoblast cell line showed good cell proliferation after 21 days of culture. The PHBHHx scaffolds demonstrated promising results in terms of cell differentiation towards an osteoblast phenotype. Copyright © 2014 John Wiley & Sons, Ltd.     

The calcification potential of human MSCs can be enhanced by interleukin‐1β in osteogenic medium

Inflammation is one of the key regulators of the repair process in bone tissues. Current data about the effect of interleukin‐1β (IL‐1β) on MSCs and osteoblasts are conflicting. We investigated the long‐term effect of IL‐1β on direct osteogenic differentiation of hMSCs in vitro. IL‐1β‐stimulated cells showed enhanced proliferation and entered maturation prior to non‐stimulated ones, as monitored by ALP activity. The process of calcification was accelerated during long‐term stimulation of hMSCs with IL‐1β. Since donor variability is a well‐known issue, we suggest a new method to illustrate global changes of a random chosen donor population through collative analysis. We further demonstrate an absorbance assay to evaluate the degree of calcification during in vitro culture of monolayer expanded hMSCs. Our findings support the importance of IL‐1β in osteogenic differentiation of hMSCs in an in vitro monolayer culture model. A new online absorbance assay is a useful method to evaluate the osteogenic differentiation of hMSCs at early stages. These findings will be helpful in optimizing predifferentiation of hMSCs in vitro for bone tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Preparation of three‐dimensional macroporous chitosan–gelatin B microspheres and HepG2‐cell culture

Chitosan–gelatin B microspheres with an open, interconnected, highly macroporous (100–200 µm) structure were prepared via a three‐step protocol combining freeze‐drying with an electrostatic and ionic cross‐linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan–gelatin B microspheres, with N‐(3‐dimethylaminopropyl)‐N′‐ethyl‐carbodiimide/N‐hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan–gelatin B microspheres to attain improved biocompatibility. Water absorption of the three‐dimensional macroporous chitosan–gelatin B microspheres (3D‐P‐CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D‐P‐CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D‐P‐CGMs was spherical, unlike that of cells cultured under traditional two‐dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D‐P‐CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D‐P‐CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D‐P‐CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.     

Efficient generation of functional hepatocyte‐like cells from menstrual blood‐derived stem cells

In recent years, the advantages of menstrual blood‐derived stem cells (MenSCs), such as minimal ethical considerations, easy access and high proliferative ability, have inspired scientists to investigate the potential of MenSCs in cell therapy of different diseases. In order to characterize the potency of these cells for future cell therapy of liver diseases, we examined the potential of MenSCs to differentiate into hepatocytes, using different protocols. First, the immunophenotyping properties and potential of MenSCs to differentiate into osteoblasts, adipocytes and chondrocytes were evaluated. Thereafter, the differentiation protocols developed by two concentrations of hepatocyte growth factor (HGF) and oncostatin M (OSM), in combination with other components in serum‐supplemented or serum‐free culture media, were also investigated. The sequential differentiation was monitored by real‐time PCR, immunostaining and functional assays. Our primary data revealed that the isolated MenSCs exhibited mesenchymal stem cell markers in parallel to OCT‐4 as an embryonic marker. Regardless of differentiation procedures, the developed cells expressed mature hepatocyte markers, such as albumin, tyrosine aminotransferase and cytokeratin‐18 at the mRNA and protein levels. They also showed functional properties of hepatocytes, including albumin secretion, glycogen storage and cytochrome P450 7A1 expression. However, the degree of differentiation was dependent on the concentrations of HGF and OSM. Indeed, omission of serum during the differentiation process caused typical improvement in hepatocyte‐specific functions. This study is a novel report demonstrating the differentiation potential of MenSCs into hepatocyte‐like cells. We recommend a complementary serum‐free differentiation protocol for enrichment of in vitro production of functional MenSC‐derived hepatocyte‐like cells that could lead to a major step toward applied stem cell therapy of chronic liver diseases. Copyright © 2013 John Wiley & Sons, Ltd.