Donor‐matched comparison of dental pulp stem cells and bone marrow‐derived mesenchymal stem cells in a rat model

Dental pulp stem cells (DPSCs) have drawn much interest for the regeneration of mineralized tissues, and several studies have compared DPSCs to bone marrow‐derived mesenchymal stem cells (BMMSCs). However, conflicting results, possibly due to donor‐associated variability, have been published and the regenerative potential of DPSCs is currently unclear. In the present study we have sought to address this problem using a donor‐matched experimental design to robustly compare the biological properties of DPSCs and BMMSCs. All experiments were performed using cells isolated from a single adult Sprague–Dawley rat. Our results show that DPSCs and BMMSCs had similar morphologies and flow cytometry profiles, were capable of forming colonies in vitro and were capable of osteogenic, chondrogenic and adipogenic differentiation. However, quantitative comparisons revealed that DPSCs had a faster population doubling time and a higher percentage of stem/progenitor cells in the population, as determined by clonogenic assays. Furthermore, while both cell populations formed mineral in vitro, DPSCs had significantly higher alkaline phosphatase activity than BMMSCs after 3 weeks in osteogenic medium. These data show several key differences between DPSCs and BMMSCs and support the possibility of using DPSCs for mineralized tissue regeneration. Copyright © 2009 John Wiley & Sons, Ltd.     

Three‐dimensional culture of mouse bone marrow cells within a porous polymer scaffold: effects of oxygen concentration and stromal layer on expansion of haematopoietic progenitor cells

To establish an ex vivo expansion method of haematopoietic progenitor cells (HPCs) and erythroid cells, three‐dimensional (3D) cultures of mouse bone marrow cells were performed, employing a porous polyvinyl formal (PVF) resin as a scaffold. In these cultures, the effects of oxygen concentration and co‐cultures with stromal cells on the expansion of HPCs and erythroid cells were investigated. When bone marrow cells were cultured under 3D conditions, HPCs and erythroid cells expanded without supplementation of exogenous cytokines, irrespective of the presence of stromal cells. On the contrary, slight expansion of HPCs or erythroid cells was observed in monolayer cultures as controls, indicating that the 3D cultures using the PVF scaffold were far better in expanding HPCs and erythroid cells than the monolayer cultures. Under hypoxic conditions, bone marrow stromal cells allowed for a 3D culture of erythroid cells and HPCs at higher cell densities compared to cultures without stromal cells, and the duration of the expansion of HPCs and erythroid cells after initiating the 3D co‐cultures was prolonged. The number of these cells increased throughout the culture period up to 3 weeks under hypoxic conditions, although the number decreased after 2 weeks under normoxic conditions. In conclusion, the 3D co‐culture method of haematopoietic cells with stromal cells under hypoxic conditions was confirmed to be effective in expanding HPCs and erythroid cells, and this method seemed to be useful for developing an ex vivo expansion method for haematopoietic cells. Copyright © 2010 John Wiley & Sons, Ltd.     

Phenotypic and functional comparison of optimum culture conditions for upscaling of bone marrow‐derived mesenchymal stem cells

Human adult bone marrow‐derived mesenchymal stem cells (MSCs) are a promising tool in the newly emerging avenue of regenerative medicine. MSCs have already been translated from basic research to clinical transplantation research. However, there is still a lack of consensus on the ideal method of culturing MSCs. Here we have compared different culture conditions of human MSCs with an attempt to preserve their characteristics and multi‐lineage differentiation potential. We compare the different basal culture media DMEM‐F12, DMEM‐high glucose (DMEM‐HG), DMEM‐low glucose (DMEM‐LG), knock‐out DMEM (DMEM‐KO) and Mesencult^® on the proliferation rate, surface markers and differentiation potentials of MSCs. At every fifth passage until the 25th passage, the differentiation potential and the presence of a panel of surface markers was observed, using flow cytometry. We also compared the characteristics of human MSCs when cultured in reduced concentrations of fetal bovine serum (FBS), knockout serum replacement (KO‐SR) and human plasma. Data indicate that the presence of serum is essential to sustain and propagate MSCs cultures. The choice of basal medium is equally important so as to preserve their characteristics and multipotent properties even after prolonged culture in vitro. With MSCs emerging as a popular tool for regenerative therapies in incurable diseases, it is essential to be able to obtain a large number of MSCs that continue to preserve their characteristics following passaging. The data reveal the optimum basal medium for prolonged culture of MSCs while retaining their ability to differentiate and hence this may be used for up‐scaling to provide sufficient numbers for transplantation. Copyright © 2009 John Wiley & Sons, Ltd.     

Accelerating bone defects healing in calvarial defect model using 3D cultured bone marrow‐derived mesenchymal stem cells on demineralized bone particle scaffold

Bone defects are usually difficult to be regenerated due to pathological states or the size of the injury. Researchers are focusing on tissue engineering approaches in order to drive the regenerative events, using stem cells to regenerate bone. The purpose of this study is to evaluate the osteogenic differentiation of bone marrow‐derived mesenchymal stem cells (BMSCs) on biologically derived Gallus gallus domesticus‐derived demineralized bone particle (GDD) sponge. The sponges were prepared by freeze‐drying method using 1, 2, and 3 wt% GDD and cross‐linked with glutaraldehyde. The GDD sponge was characterized using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared. The potential bioactivity of the sponge was evaluated by osteogenic differentiation of BMSCs using 3(4, dimethylthiazol‐2‐yl)‐2, 5‐diphenyltetrazolium bromide assay and quantifying alkaline phosphatase (ALP) activity. in vivo experiments were evaluated through a micro‐computerized tomography (μ ‐CT) and histological assays. The analysis confirmed that an increase in the concentration of the GDD in the sponge leads to a higher bone formation and deposition in rat calvarial defects. Histological assay results were in line with μ ‐CT. The results reported in this study demonstrated the potential application of GDD sponges as osteoinductor in bone tissue engineering in pathological or nonunion bone defects.     

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.     

Fabrication of porous scaffolds by three‐dimensional plotting of a pasty calcium phosphate bone cement under mild conditions

The major advantage of hydroxyapatite (HA)‐forming calcium phosphate cements (CPCs) used as bone replacement materials is their setting under physiological conditions without the necessity for thermal treatment that allows the incorporation of biological factors. In the present study, we have combined the biocompatible consolidation of CPCs with the potential of rapid prototyping (RP) techniques to generate calcium phosphate‐based scaffolds with defined inner and outer morphology. We demonstrate the application of the RP technique three‐dimensional (3D) plotting for the fabrication of HA cement scaffolds. This was realized by utilizing a paste‐like CPC (P‐CPC) which is stable as a malleable paste and whose setting reaction is initiated only after contact with aqueous solutions. The P‐CPC showed good processability in the 3D plotting process and allowed the fabrication of stable 3D structures of different geometries with adequate mechanical stability and compressive strength. The cytocompatibility of the plotted P‐CPC scaffolds was demonstrated in a cell culture experiment with human mesenchymal stem cells. The mild conditions during 3D plotting and post‐processing and the realization of the whole procedure under sterile conditions make this approach highly attractive for fabrication of individualized implants with respect to patient‐specific requirements by simultaneous plotting of biological components. Copyright © 2012 John Wiley & Sons, Ltd.     

Improved expansion of human bone marrow‐derived mesenchymal stem cells in microcarrier‐based suspension culture

Human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM‐MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier‐based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM‐MSCs in stirred suspension bioreactors using CultiSpher‐S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post‐inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM‐MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200‐fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM‐MSCs in culture, which in turn should facilitate the development of new stem cell‐based therapies. Copyright © 2012 John Wiley & Sons, Ltd.     

Recent advances in three‐dimensional bioprinting of stem cells

In spite of being a new field, three‐dimensional (3D) bioprinting has undergone rapid growth in the recent years. Bioprinting methods offer a unique opportunity for stem cell distribution, positioning, and differentiation at the microscale to make the differentiated architecture of any tissue while maintaining precision and control over the cellular microenvironment. Bioprinting introduces a wide array of approaches to modify stem cell fate. This review discusses these methodologies of 3D bioprinting stem cells. Fabricating a fully operational tissue or organ construct with a long life will be the most significant challenge of 3D bioprinting. Once this is achieved, a whole human organ can be fabricated for the defect place at the site of surgery.     

Ex vivo expansion of haematopoietic stem/progenitor cells from human umbilical cord blood on acellular scaffolds prepared from MS‐5 stromal cell line

Lineage‐specific expansion of haematopoietic stem/progenitor cells (HSPCs) from human umbilical cord blood (UCB) is desirable because of their several applications in translational medicine, e.g. treatment of cancer, bone marrow failure and immunodeficiencies. The current methods for HSPC expansion use either cellular feeder layers and/or soluble growth factors and selected matrix components coated on different surfaces. The use of cell‐free extracellular matrices from bone marrow cells for this purpose has not previously been reported. We have prepared insoluble, cell‐free matrices from a murine bone marrow stromal cell line (MS‐5) grown under four different conditions, i.e. in presence or absence of osteogenic medium, each incubated under 5% and 20% O₂ tensions. These acellular matrices were used as biological scaffolds for the lineage‐specific expansion of magnetically sorted CD34⁺ cells and the results were evaluated by flow cytometry and colony‐forming assays. We could get up to 80‐fold expansion of some HSPCs on one of the matrices and our results indicated that oxygen tension played a significant role in determining the expansion capacity of the matrices. A comparative proteomic analysis of the matrices indicated differential expression of proteins, such as aldehyde dehydrogenase and gelsolin, which have previously been identified as playing a role in HSPC maintenance and expansion. Our approach may be of value in identifying factors relevant to tissue engineering‐based ex vivo HSPC expansion, and it may also provide insights into the constitution of the niche in which these cells reside in the bone marrow. Copyright © 2012 John Wiley & Sons, Ltd.     

Biological safety of human skin‐derived stem cells after long‐term in vitro culture

The aim of this study was to investigate the biological safety of human skin‐derived mesenchymal stem cells (HSMSCs) cultured in vitro by detecting changes in karyotype, major histocompatibility complex expression and tumorigenicity. Before the 21st passage of the in vitro culture, cell surface markers were analysed by flow cytometry; major histocompatibility complex expression was detected by RT–PCR and flow cytometry. The tumorigenicity of HSMSCs was tested using SCID mice and observing changes in the injection site and pathological sections. Flow cytometry demonstrated that HSMSCs express CD73, CD105 and vimentin, but haematopoietic markers CD34, CD45 and CD19 were not expressed. Human leukocyte antigen (HLA‐I and HLA‐DR) mRNA was detected by RT–PCR; the protein expression of HLA‐I was 29.5–31.7%, but HLA‐DR protein expression was not detected in HSMSCs. The result of karyotype analysis before the 21st passage was normal and tumour formation was not detected in the mice. Taken as a whole, our results suggest that HSMSCs cultured in vitro may be safely transplanted in vivo, due to moderate expression of HLA‐I and low expression of HLA‐DR, non‐tumorigenicity and normal karyotype. Copyright © 2010 John Wiley & Sons, Ltd.     

Mechanical stimulations on human bone marrow mesenchymal stem cells enhance cells differentiation in a three‐dimensional layered scaffold

Scaffolds laden with stem cells are a promising approach for articular cartilage repair. Investigations have shown that implantation of artificial matrices, growth factors or chondrocytes can stimulate cartilage formation, but no existing strategies apply mechanical stimulation on stratified scaffolds to mimic the cartilage environment. The purpose of this study was to adapt a spraying method for stratified cartilage engineering and to stimulate the biosubstitute. Human mesenchymal stem cells from bone marrow were seeded in an alginate (Alg)/hyaluronic acid (HA) or Alg/hydroxyapatite (Hap) gel to direct cartilage and hypertrophic cartilage/subchondral bone differentiation, respectively, in different layers within a single scaffold. Homogeneous or composite stratified scaffolds were cultured for 28 days and cell viability and differentiation were assessed. The heterogeneous scaffold was stimulated daily. The mechanical behaviour of the stratified scaffolds were investigated by plane–strain compression tests. Results showed that the spraying process did not affect cell viability. Moreover, cell differentiation driven by the microenvironment was increased with loading: in the layer with Alg/HA, a specific extracellular matrix of cartilage, composed of glycosaminoglycans and type II collagen was observed, and in the Alg/Hap layer more collagen X was detected. Hap seemed to drive cells to a hypertrophic chondrocytic phenotype and increased mechanical resistance of the scaffold. In conclusion, mechanical stimulations will allow for the production of a stratified biosubstitute, laden with human mesenchymal stem cells from bone marrow, which is capable in vivo to mimic all depths of chondral defects, thanks to an efficient combination of stem cells, biomaterial compositions and mechanical loading.     

Influence of the three‐dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3

Because cell interactions play a fundamental role for cell differentiation, we investigated the expression of Pannexin 1 and Pannexin 3 in human bone marrow mesenchymal stromal cells (HBMSCs) in a three‐dimensional (3D) microenvironment provided by a polysaccharide‐based macroporous scaffold. The pannexin (Panx) family consists of three members, Panx1, Panx2, and Panx3. The roles of Panx large‐pore ion and metabolite channels are recognized in many physiological and pathophysiological scenarios, but the role of these proteins in human physiological processes is still under investigation. Our study demonstrates that HBMSCs cultured within 3D scaffolds have induced Panx1 and Panx3 expression, compared with two‐dimensional culture and that the Panx3 gene expression profile correlates with those of bone markers on mesenchymal stromal cells culture into the 3D scaffold. We showed that Panx1 is involved in the HBMSCs 3D cell–cell organization, as acting on the size of cellular aggregates, demonstrated by the use of Probenecid and the mimetic peptide 10panx1 as specific inhibitors. Inhibition of Panx3 using siRNA strategy shows to reduce the expression of osteocalcin as osteoblast‐specific marker by HBMSCs cultured in 3D conditions, suggesting a role of this Panx in osteogenesis. Moreover, we evaluated Panx1 and Panx3 expression within the cellularized scaffolds upon subcutaneous implantation in NOG (NOD/Shi‐scid/IL‐2Rγ^null) mice, where we could observe a more intense expression in the constructs than in the surrounding tissues in vivo. This study provides new insights on the expression of pannexins in HBMSCs on a 3D microenvironment during the osteogenic differentiation, in vitro and in vivo.     

Influence of in vitro biomimicked stem cell ‘niche’ for regulation of proliferation and differentiation of human bone marrow‐derived mesenchymal stem cells to myocardial phenotypes: serum starvation without aid of chemical agents and prevention of spontaneous stem cell transformation enhanced by the matrix environment

Niche appears important for preventing the spontaneous differentiation or senescence that cells undergo during in vitro expansion. In the present study, it was revealed that human bone marrow‐derived mesenchymal stem cells (hBM‐MSCs) undergo senescence‐related differentiation into the myocardial lineage in vitro without any induction treatment. This phenomenon occurred over the whole population of MCSs, much different from conventional differentiation with limited frequency of occurrence, and was accompanied by a change of morphology into large, flat cells with impeded proliferation, which are the representative indications of MSC senescence. By culturing MSCs under several culture conditions, it was determined that induction treatment with 5‐azacytidine was not associated with the phenomenon, but the serum‐starvation condition, under which proliferation is severely hampered, caused senescence progression and upregulation of cardiac markers. Nevertheless, MSCs gradually developed a myocardial phenotype under normal culture conditions over a prolonged culture period and heterogeneous populations were formed. In perspectives of clinical applications, this must be prevented for fair and consistent outcomes. Hence, the biomimetic 'niche' was constituted for hBM‐MSCs by cultivating on a conventionally available extracellular matrix (ECM). Consequently, cells on ECM regained a spindle‐shape morphology, increased in proliferation rate by two‐fold and showed decreased expression of cardiac markers at both the mRNA and protein levels. In conclusion, the outcome indicates that progression of MSC senescence may occur via myocardial differentiation during in vitro polystyrene culture, and this can be overcome by employing appropriate ECM culture techniques. Copyright © 2013 John Wiley & Sons, Ltd.     

Chondrogenesis by bone marrow‐derived mesenchymal stem cells grown in chondrocyte‐conditioned medium for auricular reconstruction

Bone marrow‐derived mesenchymal stem cells (BMSCs) can be obtained by minimally invasive means and would be a favourable source for cell‐based cartilage regeneration. However, controlling the differentiation of the BMSCs towards the desired chondrogenic pathway has been a challenge hampering their application. The major aim of the present study was to determine if conditioned medium collected from cultured auricular chondrocytes could promote chondrogenic differentiation of BMSCs. Auricular chondrocytes were isolated and grown in BMSC standard culture medium (SM) that was collected and used as chondrocyte‐conditioned medium (CCM). The BMSCs were expanded in either CCM or SM for three passages. Cells were seeded onto fibrous collagen scaffolds and precultured for 2 weeks with or without transforming growth factor‐beta 3 (TGF‐β3). After preculture, constructs were implanted subcutaneously in nude mice for 6 and 12 weeks and evaluated with real‐time polymerase chain reaction, histology, immunohistochemistry and biochemistry. Real‐time polymerase chain reaction results showed upregulation of COL2A1 in the constructs cultured in CCM compared with those in SM. After 12 weeks in vivo, abundant neocartilage formation was observed in the implants that had been cultured in CCM, with or without TGF‐β3. In contrast, very little cartilage matrix formation was observed within the SM groups, regardless of the presence of TGF‐β3. Osteogenesis was only observed in the SM group with TGF‐β3. In conclusion, CCM even had a stronger influence on chondrogenesis than the supplementation of the standard culture medium with TGF‐β3, without signs of endochondral ossification. Efficient chondrogenic differentiation of BMSCs could provide a promising alternative cell population for auricular regeneration. Copyright © 2016 John Wiley & Sons, Ltd.     

Modulation of human mesenchymal stem cell function in a three‐dimensional matrix promotes attenuation of adverse remodelling after myocardial infarction

The application of tissue engineering (TE) practices for cell delivery offers a unique approach to cellular cardiomyoplasty. We hypothesized that human mesenchymal stem cells (hMSCs) applied to the heart in a collagen matrix would outperform the same cells grown in a monolayer and directly injected for cardiac cell replacement after myocardial infarction in a rat model. When hMSC patches were transplanted to infarcted hearts, several measures for left ventricle (LV) remodelling and function were improved, including fractional area change, wall thickness, –dP/dt and LV end‐diastolic pressure. Neovessel formation throughout the LV infarct wall after hMSC patch treatment increased by 37% when compared to direct injection of hMSCs. This observation was correlated with increased secretion of angiogenic factors, with accompanying evidence that these factors enhanced vessel formation (30% increase) and endothelial cell growth (48% increase) in vitro. These observations may explain the in vivo observations of increased vessel formation and improved cardiac function with patch‐mediated cell delivery. Although culture of hMSC in collagen patches enhanced angiogenic responses, there was no effect on cell potency or viability. Therefore, hMSCs delivered as a cardiac patch showed benefits above those derived from monolayers and directly injected. hMSCs cultured and delivered within TE constructs may represent a good option to maximize the effects of cellular cardiomyoplasty. Copyright © 2011 John Wiley & Sons, Ltd.     

Enhanced recruitment and hematopoietic reconstitution of bone marrow‐derived mesenchymal stem cells in bone marrow failure by the SDF‐1/CXCR4

Aplastic anemia (AA) is a bone marrow failure disease. It is difficult to treat AA, and in addition, relapses are common because of its complex disease pathogenesis. Allogeneic bone marrow‐derived mesenchymal stem cells (BMSCs) infusion is an effective and safe treatment option for the AA patients. However, it found that BMSCs infusion in AA patients is less than 30% effective. Therefore, the key to improve the efficacy of BMSCs treatment in these patients is to enhance their homing efficiency to the target sites. Studies have shown that stromal cell‐derived factor‐1 (SDF‐1)/CXC chemokine receptor 4 (CXCR4) axis plays an important role in promoting BMSCs homing. In this study, human BMSCs were transduced with lentivirus stably expressing CXCR4‐BMSCs. Transduced BMSCs resemble normal BMSCs in many ways. Migration ability of CXCR4‐BMSCs toward SDF‐1 was increased because of the overexpression of CXCR4. In the mice with bone marrow failure, the migration and colonization ability of CXCR4‐BMSCs to the bone marrow was significantly improved as seen by IVIS imaging and FACS. The SDF‐1 level in the bone marrow failure mice was significantly higher than in the normal mice. Thus, from our study, it is clear that after CXCR4‐BMSCs were infused into mice with bone marrow failure, SDF‐1 interacted with CXCR4 receptor, leading cells to migrate and colonize to bone marrow. Because of the high SDF‐1 expression in mouse bone marrow and CXCR4 receptor expression in cells, BMSCs homing was increased.