Adipose‐ and bone marrow‐derived mesenchymal stem cells display different osteogenic differentiation patterns in 3D bioactive glass‐based scaffolds

Mesenchymal stem cells can be isolated from a variety of different sources, each having their own peculiar merits and drawbacks. Although a number of studies have been conducted comparing these stem cells for their osteo‐differentiation ability, these are mostly done in culture plastics. We have selected stem cells from either adipose tissue (ADSCs) or bone marrow (BMSCs) and studied their differentiation ability in highly porous three‐dimensional (3D) 45S5 Bioglass®‐based scaffolds. Equal numbers of cells were seeded onto 5 × 5 × 4 mm³ scaffolds and cultured in vitro, with or without osteo‐induction medium. After 2 and 4 weeks, the cell–scaffold constructs were analysed for cell number, cell spreading, viability, alkaline phosphatase activity and osteogenic gene expression. The scaffolds with ADSCs displayed osteo‐differentiation even without osteo‐induction medium; however, with osteo‐induction medium osteogenic differentiation was further increased. In contrast, the scaffolds with BMSCs showed no osteo‐differentiation without osteo‐induction medium; after application of osteo‐induction medium, osteo‐differentiation was confirmed, although lower than in scaffolds with ADSCs. In general, stem cells in 3D bioactive glass scaffolds differentiated better than cells in culture plastics with respect to their ALP content and osteogenic gene expression. In summary, 45S5 Bioglass‐based scaffolds seeded with ADSCs are well‐suited for possible bone tissue‐engineering applications. Induction of osteogenic differentiation appears unnecessary prior to implantation in this specific setting. Copyright © 2013 John Wiley & Sons, Ltd.     

Whatever their differentiation status,human progenitor derived – or mature – endothelial cells induce osteoblastic differentiation of bone marrow stromal cells

Association of the bone‐forming osteoblasts (OBs) and vascular endothelial cells (ECs) into a biomaterial composite provides a live bone graft substitute that can repair the bone defect when implanted. An intimate functional relationship exists between these cell types. This communication is crucial to the coordinated cell behaviour necessary for bone development and remodelling. Previous studies have shown that direct co‐culture of primary human osteoprogenitors (HOPs) with primary human umbilical vein endothelial cells (HUVECs) stimulates HOPs differentiation and induces tubular‐like networks. The present work aims to test the use of human bone marrow stromal cells (HBMSCs) co‐cultured with human endothelial progenitor cells in order to assess whether progenitor‐derived ECs (PDECs) could support osteoblastic differentiation as mature ECs do. Indeed, data generated from the literature by different laboratories considering these co‐culture systems appear difficult to compare. Monocultures of HUVECs, HOPs, HBMSCs (in a non‐orientated lineage), PDECs (from cord blood) were used as controls and four combinations of co‐cultures were undertaken: HBMSCs–PDECs, HBMSCs–HUVECs, HOPs–PDECs, HOPs–HUVECs with ECs (mature or progenitor) for 6 h to 7 days. At the end of the chosen co‐culture time, intracellular alkaline phosphatase (ALP) activity was detected in HOPs and HBMSCs and quantified in cell extracts. Quantitative real‐time polymerase chain reaction (qPCR) of ALP was performed over time and vascular endothelial growth factor (VEGF) was measured. After 21 days, calcium deposition was observed, comparing mono‐ and co‐cultures. We confirm that ECs induce osteoblastic differentiation of mesenchymal stem cells in vitro. Moreover, HUVECs can be replaced by PDECs, the latter being of great interest in tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Improved calvarial bone repair by hASCs engineered with Cre/loxP‐based baculovirus conferring prolonged BMP‐2 and MiR‐148b co‐expression

Repairing large calvarial bone defects remains a challenging task. Previously, it was discovered that that miR‐148b, when acting in concert with bone morphogenetic protein 2 (BMP‐2), enhanced the osteogenesis of human adipose‐derived stem cells (hASCs) and improved calvarial bone healing in nude mice. However, the molecular target of miR‐148b remained elusive. Here it is revealed that miR‐148b directly targets NOG, whose gene product (noggin) is an antagonist to BMPs and negatively regulates BMP‐induced osteogenic differentiation and bone formation. A new Cre/loxP‐based baculovirus system was employed to drive prolonged BMP‐2 and miR‐148b overexpression in hASCs, wherein the BMP‐2 overexpression induced noggin expression but the concurrent miR‐148b expression downregulated noggin, thus relieving the negative regulatory loop and ameliorating hASC osteogenesis without hindering hASC proliferation or triggering appreciable cytotoxicity. Implantation of the engineered hASCs coexpressing BMP‐2 and miR‐148b into nude mice enabled substantial repair of critical‐size calvarial bone defects (4 mm diameter) at 12 weeks post‐transplantation, filling 83% of the defect area, 75% of bone volume and restoring the bone density to 89% of the original bone density. Such superior healing effects indicate the potential of the Cre/loxP‐based baculovirus‐mediated BMP‐2/miR‐148b expression for calvarial bone repair. Copyright © 2016 John Wiley & Sons, Ltd.     

Ovine bone‐ and marrow‐derived progenitor cells and their potential for scaffold‐based bone tissue engineering applications in vitro and in vivo

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) and osteoblasts (OB) for clinical use in bone engineering. Prior to clinical application, cell based treatment concepts need to be evaluated in preclinical, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, only sheep aged > 6 years show secondary osteon formation characteristic of human bone. Osteogenic cells isolated from animals of this age group remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than OB derived from tibial compact bone as assessed in standard 2D cultures. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14^?/CD31^?/CD45^?/CD29⁺/CD44⁺/CD166⁺) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical bone related markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed > 90%. Cells displayed a spindle‐like morphology and formed interconnected networks. In contrast, when implanted subcutaneously into NOD/SCID mice, MPC presented a lower osteogenic potential than OB. In summary, this study provides a detailed characterisation of ovine MPC and OB from a bone engineering perspective and suggests that MPC and OB provide promising means for future bone disease related treatment applications. Copyright © 2010 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.     

Endothelial invasive response in a co‐culture model with physically‐induced osteodifferentiation

Manipulation of stem cells using physicochemical stimuli has emerged as an important tool in regenerative medicine. While 2D substrates with tunable elasticity have been studied for control of stem cell differentiation, we recently developed a stratified co‐culture model of angiogenesis of human mesenchymal stem cells (hMSCs) that differentiate on a tunable polydimethylsiloxane (PDMS) substrate, thereby creating a physiologic context for elasticity‐induced differentiation. Endothelial cells (EC) were cultured on top of the hMSC construct on a collagen gel to monitor network formation. Media composition influenced EC invasion due to the conditioning media, the reduction of serum and supplemental growth factors, and the addition of recombinant growth factors. Conditioned media, recombinant growth factors and direct co‐culture were compared for endothelial cell invasive response using quantitative image analysis. As anticipated, use of recombinant vascular endothelial growth factor (VEGF) induced the deepest EC invasions while direct co‐culture caused shallow invasions compared to other conditions. However, endothelial cells displayed lumen‐like morphology, suggesting that cell‐cell interaction in the co‐culture model could mimic sprouting behaviour. In summary, an engineered suitable biochemical and physical environment facilitated endothelial cells to form 3D vessel structures onto hMSCs. These structures were plated on a stiff surface known to induce osteodifferentiation of stem cells. This low cost co‐culture system, with its minimal chemical supplementation and physically controllable matrix, could potentially model in vivo potential in engineered and pre‐vascularized bone grafts. Copyright © 2012 John Wiley & Sons, Ltd.     

Co‐transplantation of Wharton's jelly mesenchymal stem cell‐derived osteoblasts with differentiated endothelial cells does not stimulate blood vessel and osteoid formation in nude mice models

A major challenge in bone tissue engineering is the lack of post‐implantation vascular growth into biomaterials. In the skeletal system, blood vessel growth appears to be coupled to osteogenesis—suggesting the existence of molecular crosstalk between endothelial cells (ECs) and osteoblastic cells. The present study (performed in two murine ectopic models) was designed to determine whether co‐transplantation of human Wharton's jelly mesenchymal stem cell‐derived osteoblasts (WJMSC‐OBs) and human differentiated ECs enhances bone regeneration and stimulates angiogenesis, relative to the seeding of WJMSC‐OBs alone. Human WJMSC‐OBs and human ECs were loaded into a silicate‐substituted calcium phosphate (SiCaP) scaffold and then ectopically implanted at subcutaneous or intramuscular sites in nude mice. At both subcutaneous and intramuscular implantation sites, we observed ectopic bone formation and osteoids composed of host cells when WJMSC‐OBs were seeded into the scaffold. However, the addition of ECs was associated with a lower level of osteogenesis, and we did not observe stimulation of blood vessel ingrowth. in vitro studies demonstrated that WJMSC‐OBs lost their ability to secrete vascular endothelial growth factor and stromal cell‐derived factor 1—including when ECs were present. In these two murine ectopic models, our cell‐matrix environment combination did not seem to be optimal for inducing vascularized bone reconstruction.     

Adipose stromal cells differentiation toward smooth muscle cell phenotype diminishes their vasculogenic activity due to induction of activin A secretion

Adipose stromal cells (ASCs) support endothelial cell (EC) vasculogenesis through paracrine and cell‐contact communications. In addition, ASCs differentiate towards the smooth muscle cell (SMC) phenotype under different stimuli, which prompted their use as a source of mural cells in fabricating small calibre vessels. How ASCs' SMC‐lineage commitment affects their subsequent communication with ECs is unknown. The vasculogenic characteristics of human ASCs in progenitor stage and after differentiation towards SMC phenotype were analysed in the present study. Exposure to transforming growth factor β1 (TGFβ₁) or activin A has induced expression of SMC markers in ASCs. Analysis performed after treatment withdrawal revealed that secretome of pre‐differentiated ASCs had a reduced potency to support EC survival and these ASCs had diminished ability to support EC vasculogenesis in vitro. Vascularization of subcutaneous implants carrying a mixture of ECs and ASCs was 50% lower when, instead of control, pre‐differentiated ASCs were used. Pre‐differentiated ASCs had an inferior mitogenic response to EC‐produced factors. Differentiation of ASCs was accompanied by upregulation of vascular endothelial growth factor and a decrease in hepatocyte growth factor (HGF) production; however, addition of HGF to the co‐culture incubation media did not improve vasculogenesis. In parallel, ASC treatment with TGFβ₁ induced secretion of activin A. Augmenting co‐culture incubation media with anti‐activin A IgG restored the ability of pre‐differentiated ASCs to support vasculogenesis to the same degree as control ASCs. The present study suggests that TGFβ₁ or activin A‐induced ASC commitment to SMC phenotype negatively affects the ability of ASCs to support EC vasculogenesis in applications based on EC and ASC co‐injection into target tissues. Copyright © 2016 John Wiley & Sons, Ltd.     

Chitosan–chondroitin sulphate nanoparticles for controlled delivery of platelet lysates in bone regenerative medicine

In this study, a new formulation of nanoparticles (NPs) based on the electrostatic interaction between chitosan and chondroitin sulphate (CH–CS NPs) is proposed for the controlled release of proteins and growth factors (GFs), specifically platelet lysates (PLs). These nanoparticulate carriers are particularly promising for protein entrapment because the interactions between the polysaccharides and the entrapped proteins mimic the interactions between chondroitin sulphate and proteins in the native extracellular matrix (ECM). Spherical non‐cytotoxic NPs were successfully produced, exhibiting high encapsulation efficiency for physiological levels of GFs and a controlled protein release profile for > 1 month. Moreover, it was also observed that these NPs can be uptaken by human adipose‐derived stem cells (hASCs), depending on the concentration of NPs in the culture medium and incubation time. This shows the versatility of the developed NPs, which, besides acting as a protein delivery system, can also be used in the future as intracellular carriers for bioactive agents, such as nucleotides. When the PL‐loaded NPs were used as a replacement of bovine serum for in vitro hASCs culture, the viability and proliferation of hASCs was not compromised. The release of PLs from CH–CS NPs also proved to be effective for the enhancement of in vitro osteogenic differentiation of hASCs, as shown by the increased levels of mineralization, suggesting not only the effective role of the delivery system but also the role of PLs as an osteogenic supplement for bone tissue engineering and regenerative medicine applications. Copyright © 2012 John Wiley & Sons, Ltd.     

In vitro differentiation of bone marrow derived porcine mesenchymal stem cells to endothelial cells

Mesenchymal stem cells (MSCs) hold potential for the regeneration of damaged tissues in cardiovascular diseases. In this study, we investigated the potential of porcine MSCs to differentiate into endothelial cells (ECs) in vitro. The cultured bone marrow‐derived cells were CD11b^–CD34^–CD44⁺CD45^–CD90⁺ and showed mesodermal lineage differentiation, which is characteristic of MSCs. The MSCs were induced to differentiate into ECs using endothelial growth medium (EGM), with and without high concentrations of VEGF (EGM + VEGF; 50 ng/ml). Endothelial basal medium (EBM) without growth factors served as the control. The EC differentiation was assessed by the presence of vWF, ability to take up acetylated LDL, in vitro angiogenesis assay, flow cytometry and qPCR of EC markers vWF, VE‐cadherin, PECAM‐1, VEGF‐R1 and VEGF‐R2 after 10 days of stimulation. Cells cultured in EGM + VEGF medium demonstrated higher amounts of DiI‐AcLDL‐positive cells and enhanced the presence of vWF (90%), VE‐Cadherin‐ (60%) and PECAM‐1 (48%)‐positive cells, than in EBM. These cells showed profuse sprouting of capillary tubes and closed polygon formation in the angiogenesis assay. There was 1.5–2‐fold increase in the mRNA expression of endothelial markers in the cells stimulated with EGM + VEGF medium when compared to control. The results demonstrate the ability of porcine MSCs to differentiate into ECs under in vitro inducing conditions. The differentiated cells would provide new options for re‐endothelialization following interventional procedures and tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.     

Alveolar bone tissue engineering in critical‐size defects of experimental animal models: a systematic review and meta‐analysis

Regeneration of large, ‘critical‐size’ bone defects remains a clinical challenge. Bone tissue engineering (BTE) is emerging as a promising alternative to autogenous, allogeneic and biomaterial‐based bone grafting. The objective of this systematic review was to answer the focused question: in animal models, do cell‐based BTE strategies enhance regeneration in alveolar bone critical‐size defects (CSDs), compared with grafting with only biomaterial scaffolds or autogenous bone? Following PRISMA guidelines, electronic databases were searched for controlled animal studies reporting maxillary or mandibular CSD and implantation of mesenchymal stem cells (MSCs) or osteoblasts (OBs) seeded on biomaterial scaffolds. A random effects meta‐analysis was performed for the outcome histomorphometric new bone formation (%NBF). Thirty‐six studies were included that reported on large‐ (monkeys, dogs, sheep, minipigs) and small‐animal (rabbits, rats) models. On average, studies presented with an unclear‐to‐high risk of bias and short observation times. In most studies, MSCs or OBs were used in combination with alloplastic mineral‐phase scaffolds. In five studies, cells were modified by ex vivo gene transfer of bone morphogenetic proteins (BMPs). The meta‐analysis indicated statistically significant benefits in favour of: (1) cell‐loaded vs. cell‐free scaffolds [weighted mean difference (WMD) 15.59–49.15% and 8.60–13.85% NBF in large‐ and small‐animal models, respectively]; and (2) BMP‐gene‐modified vs. unmodified cells (WMD 10.06–20.83% NBF in small‐animal models). Results of cell‐loaded scaffolds vs. autogenous bone were inconclusive. Overall, heterogeneity in the meta‐analysis was high (I² > 90%). In summary, alveolar bone regeneration is enhanced by addition of osteogenic cells to biomaterial scaffolds. The direction and estimates of treatment effect are useful to predict therapeutic efficacy and guide future clinical trials of BTE. Copyright © 2016 John Wiley & Sons, Ltd.     

The activin‐βA/BMP‐2 chimera AB204 is a strong stimulator of adipogenesis

Several of the bone morphogenetic proteins (BMPs) have been reported to induce white as well as brown adipogenesis. Here, we characterized the adipogenic potential of AB204, a recombinant chimeric protein of activin‐βA and BMP‐2, in in vitro, ex vivo and in vivo settings. BMP‐2 is generally known to promote adipogenesis. When compared with BMP‐2, which previously showed varying degrees of adipogenesis, AB204 displayed superior in vitro adipogenic differentiation of mouse 3 T3‐L1 pre‐adipocytes and human adipose‐derived stem cells (hASCs). Surprisingly, implantation of hASCs, preconditioned with AB204 for as short a time as 48 h, into the subcutaneous space of athymic nude mice effectively produced fat pads, but not with BMP‐2. When BMP‐2 and AB204 were injected intraperitoneally, AB204 promoted dramatic systemic adipogenesis of C57BL/6 mice on a high‐fat diet very effectively. The results implicate the novel clinical potential of AB204, including induction of fat tissue ex vivo or in vivo for tissue re‐engineering and regenerative medicinal purposes, more than any known natural protein ligand. Copyright © 2015 John Wiley & Sons, Ltd.     

Effects of resveratrol on enrichment of adipose‐derived stem cells and their differentiation to osteoblasts in two‐and three‐dimensional cultures

The goal of this study was to develop a method for increasing the yield of multipotent adipose‐derived mesenchymal stem cells (ASCs) and osteoprogenitor cells (OPCs) from subcutaneous fat. After removing mature adipocytes and haematopoietic cells from rat inguinal fat, ASCs in the remaining cell population were verified by their attachment to plastic, surface marker profile (CD271⁺, CD73⁺ and CD45^–) and ability to differentiate into adipocytes, chondrocytes and osteoblasts. OPCs were defined as E11⁺ and OCN⁺. Adherent cells were cultured in growth medium (GM) or osteogenic medium (OM) and treated with resveratrol (0, 12.5, and 25 µ m ) for 7 days; ASCs and OPCs were assessed by flow cytometry. Osteogenic potential was determined in two‐dimensional (2D) cultures as a function of alkaline phosphatase‐specific activity and osteocalcin production. In addition, cells were seeded onto three‐dimensional (3D) poly‐ε‐caprolactone scaffolds and cultured under dynamic conditions; mineralization was quantified by micro‐CT at 4, 8 and 12 weeks. Resveratrol increased the percentage of ASCs in the population (population%) and number of ASCs in both GM and OM, but increased only the number of OPCs in GM. In both media types resveratrol increased alkaline phosphatase activity and osteocalcin levels. In 3D cultures, resveratrol‐treated cells significantly increased mineralized matrix volume at early time points. Resveratrol exerted a biphasic effect on adherent cells by enriching the ASC and OPC populations and enhancing osteogenic differentiation. Resveratrol pretreatment induced more mineralization at earlier time points and represents a clinically viable technique for orthopaedic and dental applications for autologous stem cell therapy. Copyright © 2012 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.     

Differential bone‐forming capacity of osteogenic cells from either embryonic stem cells or bone marrow‐derived mesenchymal stem cells

For more than a decade, human mesenchymal stem cells (hMSCs) have been used in bone tissue‐engineering research. More recently some of the focus in this field has shifted towards the use of embryonic stem cells. While it is well known that hMSCs are able to form bone when implanted subcutaneously in immune‐deficient mice, the osteogenic potential of embryonic stem cells has been mainly assessed in vitro. Therefore, we performed a series of studies to compare the in vitro and in vivo osteogenic capacities of human and mouse embryonic stem cells to those of hMSCs. Embryonic and mesenchymal stem cells showed all characteristic signs of osteogenic differentiation in vitro when cultured in osteogenic medium, including the deposition of a mineralized matrix and expression of genes involved in osteogenic differentiation. As such, based on the in vitro results, osteogenic ES cells could not be discriminated from osteogenic hMSCs. Nevertheless, although osteogenic hMSCs formed bone upon implantation, osteogenic cells derived from both human and mouse embryonic stem cells did not form functional bone, indicated by absence of osteocytes, bone marrow and lamellar bone. Although embryonic stem cells show all signs of osteogenic differentiation in vitro, it appears that, in contrast to mesenchymal stem cells, they do not possess the ability to form bone in vivo when a similar culture method and osteogenic differentiation protocol was applied. Copyright © 2010 John Wiley & Sons, Ltd.     

Three‐dimensional printed polycaprolactone‐based scaffolds provide an advantageous environment for osteogenic differentiation of human adipose‐derived stem cells

The capacity of bone grafts to repair critical size defects can be greatly enhanced by the delivery of mesenchymal stem cells (MSCs). Adipose tissue is considered the most effective source of MSCs (ADSCs); however, the efficiency of bone regeneration using undifferentiated ADSCs is low. Therefore, this study proposes scaffolds based on polycaprolactone (PCL), which is widely considered a suitable MSC delivery system, were used as a three‐dimensional (3D) culture environment promoting osteogenic differentiation of ADSCs. PCL scaffolds enriched with 5% tricalcium phosphate (TCP) were used. Human ADSCs were cultured in osteogenic medium both on the scaffolds and in 2D culture. Cell viability and osteogenic differentiation were tested at various time points for 42 days. The expression of RUNX2, collagen I, alkaline phosphatase, osteonectin and osteocalcin, measured by real‐time polymerase chain reaction was significantly upregulated in 3D culture. Production of osteocalcin, a specific marker of terminally differentiated osteoblasts, was significantly higher in 3D cultures than in 2D cultures, as confirmed by western blot and immunostaining, and accompanied by earlier and enhanced mineralization. Subcutaneous implantation into immunodeficient mice was used for in vivo observations. Immunohistological and micro‐computed tomography analysis revealed ADSC survival and activity toward extracellular production after 4 and 12 weeks, although heterotopic osteogenesis was not confirmed – probably resulting from insufficient availability of Ca/P ions. Additionally, TCP did not contribute to the upregulation of differentiation on the scaffolds in culture, and we postulate that the 3D architecture is a critical factor and provides a useful environment for prior‐to‐implantation osteogenic differentiation of ADSCs. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Reconstruction of hepatic stellate cell‐incorporated liver capillary structures in small hepatocyte tri‐culture using microporous membranes

In liver sinusoids, hepatic stellate cells (HSCs) locate the outer surface of microvessels to form a functional unit with endothelia and hepatocytes. To reconstruct functional liver tissue in vitro, formation of the HSC‐incorporated sinusoidal structure is essential. We previously demonstrated capillary formation of endothelial cells (ECs) in tri‐culture, where a polyethylene terephthalate (PET) microporous membrane was intercalated between the ECs and hepatic organoids composed of small hepatocytes (SHs), i.e. hepatic progenitor cells, and HSCs. However, the high thickness and low porosity of the membranes limited heterotypic cell–cell interactions, which are essential to form HSC–EC hybrid structures. Here, we focused on the effective use of the thin and highly porous poly( d , l ‐lactide‐co‐glycolide) (PLGA) microporous membranes in SH–HSC–EC tri‐culture to reconstruct the HSC‐incorporated liver capillary structures in vitro. First, the formation of EC capillary‐like structures was induced on Matrigel‐coated PLGA microporous membranes. Next, the membranes were stacked on hepatic organoids composed of small SHs and HSCs. When the pore size and porosity of the membranes were optimized, HSCs selectively migrated to the EC capillary‐like structures. This process was mediated in part by platelet‐derived growth factor (PDGF) signalling. In addition, the HSCs were located along the outer surface of the EC capillary‐like structures with their long cytoplasmic processes. In the HSC‐incorporated capillary tissues, SHs acquired high levels of differentiated functions, compared to those without ECs. This model will provide a basis for the construction of functional, thick, vascularized liver tissues in vitro. Copyright © 2012 John Wiley & Sons, Ltd.     

Human adipose‐derived cells can serve as a single‐cell source for the in vitro cultivation of vascularized bone grafts

Orthopaedic surgery often requires bone grafts to correct large defects resulting from congenital defects, surgery or trauma. Great improvements have been made in the tissue engineering of bone grafts. However, these grafts lack the vascularized component that is critical for their survival and function. From a clinical perspective, it would be ideal to engineer vascularized bone grafts starting from one single‐cell harvest obtained from the patient. To this end, we explored the potential of human adipose‐derived mesenchymal stem cells (hASCs) as a single‐cell source for osteogenic and endothelial differentiation and the assembly of bone and vascular compartments within the same scaffold. hASCs were encapsulated in fibrin hydrogel as an angioinductive material for vascular formation, combined with a porous silk fibroin sponge to support osteogenesis, and subjected to sequential application of growth factors. Three strategies were evaluated by changing spatiotemporal cues: (a) induction of osteogenesis prior to vasculogenesis; (b) induction of vasculogenesis prior to osteogenesis; or (c) simultaneous induction of osteogenesis and vasculogenesis. By 5 weeks of culture, bone‐like tissue development was evidenced by the deposition of bone matrix proteins, alkaline phosphatase activity and calcium deposition, along with the formation of vascular networks, evidenced by endothelial cell surface markers, such as CD31 and von Willebrand factor, and morphometric analysis. Most robust development of the two tissue compartments was achieved by sequential induction of osteogenesis followed by the induction of vasculogenesis. Taken together, the collected data strongly support the utility of hASCs as a single‐cell source for the formation of vascularized bone tissue. Copyright © 2012 John Wiley & Sons, Ltd.