Repair of mandibular defects using MSCs-seeded biodegradable polyester porous scaffolds

PLLA, PLA-PEG and PLGA porous scaffolds with pore size ranging from 100 to 250 microm and porosity over 85% were fabricated by a solution-casting/salt-leaching method. The porous structure and porosity of the scaffold were mainly dependent on volume fraction and size of the porogens of NaCl particles. The effects of the polymeric materials on the cell culture behavior and bone formation in vitro in their scaffolds were studied. In vitro cell culture in the scaffolds of the three polymers demonstrated that mesenchymal stem cells (MSCs) had a good adhesion and spread. The composite matrixes cultured for several days possessed preliminary functions of tissue-engineering bone, with signs of the calcium knur formation and the expression of osteocalcin and collagen I in mRNA, especially that of PLA-PEG and PLGA. These cell-loaded porous scaffolds showed effective repair of mandibular defect of rabbits in vivo. Contrastive experiments demonstrated that the MSCs/PLGA scaffold owned better ability facilitating for the MSCs proliferation, differentiation and defect repair. These composite scaffolds can be a potential effective tool for treating mandibular and other bone defects.     

Influence of decellularized matrix derived from human mesenchymal stem cells on their proliferation, migration and multi-lineage differentiation potential

Developing biomaterials to promote stem cell proliferation and differentiation is a critical requirement in tissue engineering and regeneration. Extracellular matrix (ECM) derived from mesenchymal stem cells (MSCs) has recently been shown to be able to maintain the differentiation potential of MSCs during culture expansion and to restore the activities of aging MSCs, suggesting that MSC ECM (MECM) may be a suitable culture substrate to enhance the bioactivity of biomaterial scaffolds for MSCs. This investigation aims to characterize the biological nature and specificity of the influence of the MECM on MSCs. Native ECM produced by human MSC in vitro was extracted in urea, and the residual pellet was further processed with pepsin digestion (denoted as U-MECM and HP-MECM, respectively). The MECM products were then coated as a substrate on standard tissue culture plastic, and the behavior of MSCs seeded on the coated surfaces was studied. Our results showed that U-MECM coating dramatically accelerated MSC proliferation, attachment, spread, migration and multi-lineage differentiation (i.e., osteogenesis and adipogenesis), compared to collagen type I and HP-MECM coating. Non-collagenous proteins are likely the bioactive components in U-MECM, as MSCs cultured on collagen type I and HP-MECM showed similar biological activities, and collagen type I appeared to be the major protein components remaining in HP-MECM based on SDS-PAGE. These findings support the biological utility of MECM in the formulation of biomaterial scaffolds to enhance MSC bioactivities, including proliferation, migration and multi-lineage differentiation, for tissue regeneration applications.     

Enhancement of chondrogenic differentiation of human articular chondrocytes by biodegradable polymers.

Biodegradable polymers are attractive candidates for chondrocyte embedding and transplantation in cartilage tissue engineering. In an attempt to determine the effects of a variety of biodegradable materials on cartilage proliferation and extracellular matrix production, poly-L-lactic acid (PLLA) with a molecular weight of 5,000, polyglycolic acid (PGA) with a molecular weight of 3,000, and copolymer of poly(L-lactic acid-glycolic acid) 50:50 (PLGA) with a molecular weight of 5,000, were dissolved in DMSO and added into the medium for 4 weeks in in vitro high-density micromass culture of multiplied human articular chondrocytes (HAC). PLLA with a molecular weight of 270,000 (PLAO3) was used as thin film. Cell proliferation and differentiation in these biomaterials were compared with tissue culture polystyrene (TCPS) as a control. Alamar blue and alcian blue staining were carried out to determine the chondrocyte proliferation and differentiation, respectively. Samples exposed to these biomaterials promoted cell proliferation in the range of 86-105% of the control proliferation, and a slight but significant increase in cell proliferation was noted only in the culture exposed to PLGA. The sample exposed to PGA elicited a significant 3.7-fold higher (p < 0.01) cell differentiation than controls and was significantly higher than that of the samples exposed to PLLA, PLAO3, and PLGA. After 4 weeks of culture, the cell differentiation from most to least was in the following order PGA > PLAO3 > PLGA = PLLA > Cont. = DMSO. Chondrocyte differentiation of the samples exposed to various biomaterials were significantly higher compared with controls. Thus, serially passage chondrocytes are competent for cell growth and quantifiable matrix production, and biodegradable polymers, especially PGA, hold promise as suitable substrates for scaffolding materials for human cartilage tissue engineering.     

Analysis of mesenchymal stem cells grown on a three-dimensional HYAFF 11-based prototype ligament scaffold

Ligaments are complex structures that maintain the mechanical stability of the joint. Healing of injured ligaments involves the interactions of different cell types, local cellular environment, and the use of devices. To gain new information on the complex interactions between mesenchymal stem cells (MSCs) and a specific hyaluronan-based prototype scaffold (HYAFF, useful for ligament tissue engineering, short time-course experiments were performed to analyze the proliferation, vitality, and phenotype of MSCs grown on the scaffold. MSC proliferation was analyzed using the MTT test, during the early time points (2, 4, 6, days). Viability was assessed using calcein/acetyloxymethylester immunofluorescence dye and confocal microscopy analysis. Hyaluronic acid receptor (CD44), typical matrix ligament proteins (collagen type I, type III, laminin, fibronectin, actin), and chondrogenic/osteogenic markers (collagen type II and bone sialoprotein) were evaluated by immunohistochemistry. Our data demonstrated that MSC growth and viability were cell density-dependent. MSCs completely wrapped the fibers of the scaffold, expressed CD44, collagen type I, type III, laminin, fibronectin, and actin, and were negative to collagen type II and bone sialoprotein. These data demonstrate that MSCs survive well in the hyaluronan-based prototype ligament scaffold, as assessed after 2 days from seeding, and express CD44, a receptor important for scaffold interaction, and proteins responsible for the functional characteristics of the ligaments.     

In vitro differentiation of human mesenchymal stem cells on Ti6Al4V surfaces

Long-term stability of arthroplasty prosthesis depends on the integration between the bone tissue and the implanted biomaterials, which requires the contribution of osteoblastic precursors and their continuous differentiation into the osteoblastic phenotype. Classically, these interactions are tested in vitro using mesenchymal stem cells (MSCs) isolated and ex vivo expanded from bone marrow aspirates. Human adipose tissue-derived stromal cells (AMSCs) may be a more convenient source of MSCs, according to their abundance and accessibility, but no data are available on their in vitro interactions with hard biomaterials. The aim of this work is to compare the osteogenic potential of human AMSCs and bone marrow-derived MSCs (BMMSCs) and to evaluate their response to Ti6Al4V alloy in terms of adhesion, proliferation and differentiation features, using the human osteosarcoma cell line SaOS-2 for comparison. The overall results showed that AMSCs have the same ability to produce bone matrix as BMMSCs and that Ti6Al4V surfaces exhibit an osteoinductive action on AMSCs, promoting their differentiation into functional osteoblasts and increasing bone formation. In conclusion, adipose tissue is a promising autologous source of osteoblastic cells with important clinical implications for bone tissue engineering.     

Enhanced differentiation of mesenchymal stem cells co-cultured with ligament fibroblasts on gelatin/silk fibroin hybrid scaffold

The differentiation of mesenchymal stem cells (MSCs) towards fibroblasts is a crucial issue in ligament tissue engineering. This study aims to investigate the feasibility of using co-culture system to induce the differentiation of MSCs for constructing the tissue-engineered ligament in vitro. A kind of silk cable-reinforced gelatin/silk fibroin hybrid scaffold was used to provide three-dimensional (3-D) culture environments for MSCs. The 3-D co-culture system was set up by culturing MSCs/scaffold and ligament fibroblasts in the transwell insert and lower chamber, respectively. The regulatory effects of fibroblasts on MSCs were determined. After 2 weeks of co-culture the MSCs showed faster proliferation and higher DNA content compared with MSCs non-co-cultured. The MSCs were distributed uniformly throughout the scaffold and showed good viability. The collagen production also increased significantly with culture time. The MSCs in co-culture system were proved to differentiate into ligament fibroblasts by expressing ligament extra-cellular matrix (ECM)-specific genes including collagen I, collagen III, and tenascin-C in mRNA and protein level. The immunohistochemistry staining also confirmed the synthesis of key ligament ECM components. This study reveals that specific regulatory signals released from fibroblasts in 3-D co-culture system can enhance the differentiation of MSCs for ligament tissue engineering.     

Functionalization of poly(L-lactide) nanofibrous scaffolds with bioactive collagen molecules

The utilization of electrospun biodegradable scaffolds by fine-tuning their biofunctionalities through a simple mixing method was demonstrated in this study. Poly(L-lactide) (PLLA)-based scaffolds containing small amounts of bioactive collagen type I molecules were investigated for enhancements in cellular behavior. Electron microscopy revealed no topological alterations of the fibers in the collagen/PLLA scaffolds when compared with pure PLLA scaffolds. Cell attachment after 24 h was robust on collagen/PLLA scaffolds, with cytoskeletal analysis showing that the attached cells were aligned along the fibers assuming a spindle-shape appearance. Despite these morphological differences, gene expression analyses revealed no apparent alterations in mRNA levels of four genes involved in cell attachment across the various scaffolds. Although cell proliferation was not adversely affected, there were clear differences in cell penetration; after 1 week, cells migrated through 32 and 85% of PLLA and collagen/PLLA scaffolds, respectively. Mineralization of primary calvaria osteoblasts provided further evidence that collagen-containing electrospun PLLA scaffolds could sustain cell differentiation. Overall, the inclusion of collagen type I in even miniscule amounts (<1 wt %) within electrospun PLLA scaffolds could effectively modulate certain aspects of cellular behavior.     

Cellular and molecular events during chondrogenesis of human mesenchymal stromal cells grown in a three-dimensional hyaluronan based scaffold

Mesenchymal stromal cells (MSCs) seem to be a good alternative to chondrocytes for cartilage regeneration. To obtain new information on the sequence of cellular and molecular events during in vitro chondrogenic differentiation we analysed MSCs on a widely used hyaluronic acid biomaterial (Hyaff-11). Cellular differentiation was induced using two different concentrations of TGFbeta1 (10 and 20 ng/ml) and the process was analysed at different time points (24 h, and 7, 14, 21 and 28 days) using techniques of light and electron microscopy, real-time PCR and immunohistochemistry. We found that without TGFbeta MSCs did not survive while in the presence of TGFbeta the cells significantly proliferated from day 7 until day 28. Light and electron microscopy showed that TGFbeta at 20 ng/ml better induced the formation of cartilage-like tissue. Real-time PCR showed an increased expression of collagen type II, IX and aggrecan associated to a down-regulation of collagen type I. Immunohistochemical analysis confirmed that collagen type I was down-modulated while collagen type II increased from day 14 to day 28. These data clearly showed that higher concentrations of TGFbeta (20 ng/ml) induce chondrogenesis of MSCs on Hyaff-11 scaffold better than 10 ng/ml of TGFbeta. This process is characterized by a sequence of cellular and molecular events that deal with the in vitro formation of a cartilage-like tissue.     

Controlling the fibroblastic differentiation of mesenchymal stem cells via the combination of fibrous scaffolds and connective tissue growth factor

Controlled differentiation of multi-potent mesenchymal stem cells (MSCs) into vocal fold-specific, fibroblast-like cells in vitro is an attractive strategy for vocal fold repair and regeneration. The goal of the current study was to define experimental parameters that can be used to control the initial fibroblastic differentiation of MSCs in vitro. To this end, connective tissue growth factor (CTGF) and micro-structured, fibrous scaffolds based on poly(glycerol sebacate) (PGS) and poly(?-caprolactone) (PCL) were used to create a three-dimensional, connective tissue-like microenvironment. MSCs readily attached to and elongated along the microfibers, adopting a spindle-shaped morphology during the initial 3 days of preculture in an MSC maintenance medium. The cell-laden scaffolds were subsequently cultivated in a conditioned medium containing CTGF and ascorbic acids for up to 21 days. Cell morphology, proliferation, and differentiation were analyzed collectively by quantitative PCR analyses, and biochemical and immunocytochemical assays. F-actin staining showed that MSCs maintained their fibroblastic morphology during the 3 weeks of culture. The addition of CTGF to the constructs resulted in an enhanced cell proliferation, elevated expression of fibroblast-specific protein-1, and decreased expression of mesenchymal surface epitopes without markedly triggering chondrogenesis, osteogenesis, adipogenesis, or apoptosis. At the mRNA level, CTGF supplement resulted in a decreased expression of collagen I and tissue inhibitor of metalloproteinase 1, but an increased expression of decorin and hyaluronic acid synthesase 3. At the protein level, collagen I, collagen III, sulfated glycosaminoglycan, and elastin productivity was higher in the conditioned PGS-PCL culture than in the normal culture. These findings collectively demonstrate that the fibrous mesh, when combined with defined biochemical cues, is capable of fostering MSC fibroblastic differentiation in vitro.     

Three-dimensional culture of differentiating marrow stromal osteoblasts in biomimetic poly(propylene fumarate-co-ethylene glycol)-based macroporous hydrogels

This study assesses the ability of biomimetic poly(propylene fumarate-co-ethylene glycol)-based hydrogels to sustain the differentiation of marrow stromal cells (MSCs) to the osteoblastic phenotype and to produce a mineralized matrix in vitro. Macroporous hydrogels based on poly(propylene fumarate-co-ethylene glycol) with and without covalently linked RGD cell-adhesive peptide were synthesized and seeded with rat MSCs suspended in media or in a type I collagen solution. Cells suspended in media were found to adhere to RGD-modified but not to unmodified hydrogels. Cells suspended in a collagen solution were entrapped after collagen gelation and proliferated independent of the peptide modification of the hydrogel. Hydrogel modification with RGD peptide was sufficient to allow for the adhesion and differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture supplements. MSCs seeded with a collagen gel onto RGD-modified macroporous hydrogels after 28 days of culture showed a significant increase in cell numbers, from 15,200 +/- 2,000 to 208,600 +/- 69,700 cells (p < 0.05). Moreover, significant calcium deposition was apparent after 28 days of culture in RGD-modified hydrogels for cells suspended in a collagen gel in comparison to cells suspended in media, 3.47 +/- 0.26 compared to 0.82 +/- 0.20 mg Ca(2+) per scaffold (p < 0.05). Confocal microscopy revealed that MSCs suspended in a collagen gel and cultured on RGD-modified hydrogels for 28 days were adhered to the surface of the hydrogel while MSCs suspended in a collagen gel and cultured on unmodified hydrogels were located within the pores of and not in direct contact with the hydrogel surface. The results demonstrate that these biomimetic hydrogels facilitate the adhesion and support the differentiation of MSCs to the osteoblastic phenotype in the presence of osteogenic culture media.     

Effect of rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins in vitro

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation because it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins by human bone-derived cells (HBDCs) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, two materials with a crystalline phase Ca(2)KNa(PO(4))(2) and with a small amorphous portion containing either magnesium potassium phosphate (material denominated GB14) or silica phosphate (material denominated GB9), and a calcium phosphate bone cement (material denominated Biocement D). HBDCs were grown on the substrata for 3, 7, 14, and 21 days, counted, and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All substrates supported continuous cellular growth for 21 days. In the presence of GB14 and Biocement D specimens cell proliferation was reduced and cell differentiation increased. At day 21, the greatest number of cells was found on GB9 expressing significantly higher levels of bone-related proteins than cells grown on all other surfaces. Because all novel materials facilitated the expression of the osteoblastic phenotype at least as much as TCP and the polystyrene control, these biomaterials can be regarded as excellent candidate bone substitute materials. GB9 induced the highest proliferation and cellular differentiation after 21 days of incubation, suggesting that this material may possess a higher potency for enhancing osteogenesis than TCP.     

The effect of mesenchymal stem cell osteoblastic differentiation on the mechanical properties of engineered bone-like tissue

Mesenchymal stem cells (MSCs) can give rise to osteoblasts and have therefore been suggested as a cell source for bone engineering. Here we hypothesized that MSC osteoblastic differentiation and maturation can be supported by three-dimensional cultures in collagen hydrogels (hydrogel culture) to ultimately give rise to mechanically robust bone-like tissue. We first compared the osteoblastic differentiation efficiency of MSCs using osteoinductive supplements (β-glycerophosphate, vitamin C, and dexamethasone) in a hydrogel culture and in a two-dimensional culture (2D culture) by assessing surrogate parameters for osteoblastic differentiation, including osteocalcin (OC) secretion and calcium (Ca) deposition. We next constructed ring-shaped bone-like tissues using MSCs in the hydrogel cultures, and assessed their mechanical (strain-strain analysis), biochemical/molecular (OC secretion, Ca deposition, and Runx2/osterix mRNA levels), and morphological (von Kossa staining) properties. OC secretions and Ca depositions were significantly higher in the hydrogel cultures than those in the 2D cultures, suggesting better osteoblastic differentiation and maturation in the hydrogel cultures. Collagen hydrogel-based ring-shaped bone-like tissues conditioned with osteoinductive supplements developed enhanced biomechanical properties, including high tissue stiffness and ultimate burst strength, superior molecular/biochemical properties, and morphological signs typically found in mineralized bone. These results may be exploited not only to generate bioartificial bone, but also to elucidate the basic mechanisms of bone physiology.     

Human heart, spleen, and perirenal fat-derived mesenchymal stem cells have immunomodulatory capacities

Mesenchymal stem cells (MSCs) have important tissue repair functions and show potent immunosuppressive capacities in vitro. Although usually isolated from the bone marrow, MSCs have been identified in other tissues, including the skin and liver. In the present study, we isolated and characterized MSCs from human heart, spleen, and perirenal adipose tissue. MSCs from these different tissue sites were similar to those derived from bone marrow in that they expressed comparable levels of the cell-surface markers CD90, CD105, CD166, and HLA class I, were negative for CD34, CD45, HLA class II, CD80, and CD86 expression, and were capable of osteogenic and adipogenic differentiation. Like bone marrow-derived MSCs, MSCs from these different tissue sources inhibited the proliferation of alloactivated peripheral blood mononuclear cells (PBMCs), giving 85%, 79%, 79%, and 81% inhibition, respectively. Also in line with bone marrow-derived MSCs they inhibited proliferative responses of PBMCs to phytohemagglutinin, a nonspecific stimulator of lymphocyte proliferation, and reduced-memory T lymphocyte responses to tetanus toxoid. The results of this study demonstrate that MSCs from various tissues have similar immunophenotypes, in vitro immunosuppressive properties, and differentiation potential.     

The growth and morphological behavior of salivary epithelial cells on matrix protein-coated biodegradable substrata

The purpose of this study was to examine the growth and morphology of a salivary epithelial cell line (HSG) in vitro on several biodegradable substrata as an important step toward developing an artificial salivary gland. The substrates examined were poly-L-lactic acid (PLLA), polyglycolic acid (PGA), and two co-polymers, 85% and 50% PLGA, respectively. The substrates were formed into 20- to 25-mm disks, and the cells were seeded directly onto the polymers or onto polymers coated with specific extracellular matrix proteins. The two copolymer substrates became friable over time in aqueous media and proved not useful for these experiments. The purified matrix proteins examined included fibronectin (FN), laminin (LN), collagen I, collagen IV, and gelatin. In the absence of preadsorbed proteins, HSG cells did not attach to the polymer disks. The cells, in general, behaved similarly on both PLLA and PGA, although optimal results were obtained consistently in PLLA. On FN-coated PLLA disks, HSG cells were able to form a uniform monolayer, which was dependent on time and FN concentration. Coating of disks with LN, collagen I, and gelatin also promoted monolayer growth. This study defines the conditions necessary for establishing a monolayer organization of salivary epithelial cells with rapid proliferation on a biodegradable substrate useful for tissue engineering.     

Significance of nano- and microtopography for cell-surface interactions in orthopaedic implants

Cell-surface interactions play a crucial role for biomaterial application in orthopaedics. It is evident that not only the chemical composition of solid substances influence cellular adherence, migration, proliferation and differentiation but also the surface topography of a biomaterial. The progressive application of nanostructured surfaces in medicine has gained increasing interest to improve the cytocompatibility and osteointegration of orthopaedic implants. Therefore, the understanding of cell-surface interactions is of major interest for these substances. In this review, we elucidate the principle mechanisms of nano- and microscale cell-surface interactions in vitro for different cell types onto typical orthopaedic biomaterials such as titanium (Ti), cobalt-chrome-molybdenum (CoCrMo) alloys, stainless steel (SS), as well as synthetic polymers (UHMWPE, XLPE, PEEK, PLLA). In addition, effects of nano- and microscaled particles and their significance in orthopaedics were reviewed. The significance for the cytocompatibility of nanobiomaterials is discussed critically.     

Repair of mandibular defects using MSCs-seeded biodegradable polyester porous scaffolds

PLLA, PLA-PEG and PLGA porous scaffolds with pore size ranging from 100 to 250 μm and porosity over 85% were fabricated by a solution-casting/salt-leaching method. The porous structure and porosity of the scaffold were mainly dependent on volume fraction and size of the porogens of NaCl particles. The effects of the polymeric materials on the cell culture behavior and bone formation in vitro in their scaffolds were studied. In vitro cell culture in the scaffolds of the three polymers demonstrated that mesenchymal stem cells (MSCs) had a good adhesion and spread. The composite matrixes cultured for several days possessed preliminary functions of tissue-engineering bone, with signs of the calcium knur formation and the expression of osteocalcin and collagen I in mRNA, especially that of PLA-PEG and PLGA. These cell-loaded porous scaffolds showed effective repair of mandibular defect of rabbits in vivo. Contrastive experiments demonstrated that the MSCs/PLGA scaffold owned better ability facilitating for the MSCs proliferation, differentiation and defect repair. These composite scaffolds can be a potential effective tool for treating mandibular and other bone defects.     

Probing the osteoinductive effect of calcium phosphate by using an in vitro biomimetic model

The use of calcium phosphate (CaP)-based carriers in bone engineering is a promising approach to induce in vivo bone formation. However, the exact mechanism of osteoinduction by CaP is not known. Here, by mimicking the in vivo Ca(2+) and P(i)-enriched environment in an in vitro model, we assessed the effects of these ions on human periosteum-derived cells. We observed a significant Ca(2+) and P(i)-induced cell proliferation, which was found to be through the modulation of cell cycle progression, in a dose- and time-dependent manner. In addition, Ca(2+), P(i), and combined Ca-P upregulated osteogenic gene expression in a dose- and time-dependent manner. Encouragingly, both ions administered individually or in combination persistently and dose dependently upregulated bone morphogenetic protein-2 gene expression. This suggested a potential osteoinductive effect through an autonomous activation of the bone morphogenetic protein signaling pathway by released Ca(2+) and P(i), which may serve as an autocrine/paracrine osteoinduction loop that drives the cellularized CaP constructs toward effective bone formation in vivo. In conclusion, through an in vitro biomimetic model, we have partially probed the roles of the released Ca(2+) and P(i) on the osteoinductivity of CaP-based biomaterials.     

Flow Perfusion Enhances the Calcified Matrix Deposition of Marrow Stromal Cells in Biodegradable Nonwoven Fiber Mesh Scaffolds

In this study, we report on the ability of resorbable poly(L-lactic acid) (PLLA) nonwoven scaffolds to support the attachment, growth, and differentiation of marrow stromal cells (MSCs) under fluid flow. Rat MSCs were isolated from young male Wistar rats and expanded using established methods. The cells were then seeded on PLLA nonwoven fiber meshes. The PLLA nonwoven fiber meshes had 99% porosity, 17 m fiber diameter, 10 mm scaffold diameter, and 1.7-mm thickness. The nonwoven PLLA meshes were seeded with a cell suspension of 5 × 10⁵ cells in 300 l, and cultured in a flow perfusion bioreactor and under static conditions. Cell/polymer nonwoven scaffolds cultured under flow perfusion had significantly higher amounts of calcified matrix deposited on them after 16 days of culture. Microcomputed tomography revealed that the in vitro generated extracellular matrix in the scaffolds cultured under static conditions was denser at the periphery of the scaffold while in the scaffolds cultured in the perfusion bioreactor the extracellular matrix demonstrated a more homogeneous distribution. These results show that flow perfusion accelerates the proliferation and differentiation of MSCs, seeded on nonwoven PLLA scaffolds, toward the osteoblastic phenotype, and improves the distribution of the in vitro generated calcified extracellular matrix.     

Tumor-conditioned mesenchymal stem cells display hematopoietic differentiation and diminished influx of Ca2+

Mesenchymal stem cells (MSCs) that are present in many adult tissues can generate new cells either continuously or in response to injury/cancer. An increasing number of studies demonstrated that MSCs have the ability to differentiate into cells of mesodermal origin and transdifferentiate into cells such as hepatocytes, neural cells. There has been growing interest in the application of MSCs to cancer therapy. The relationship between MSCs and cancer cells remains highly controversial. In this study, we analyzed the interaction of bone marrow-derived MSCs and cancer cells by cell-cell contact and transwell culture system. The flow cytometry and real-time polymerase chain reaction showed that after coculture of MSCs and cancer cells, MSCs displayed the hematopoietic cell markers such as CD34, CD45, and CD11b. The CD68, MRCI, and CSF1R were dramatically upregulated after coculture. The cytokine array showed that MSCs after coculture secreted monokines and chemokines much more than that of intact MSCs. The MSCs under tumor conditions were responsive to stimulation with lipopolysaccharide by cytokines release. The tumor-conditioned MSCs showed phagocytic ability and enhanced release of nitric oxide, which are the characteristics of macrophages. Calcium ion is an important intracellular messenger responsible for differentiation and gene expression regulations. The influx of Ca(2+) into MSCs was obviously reduced after coculture. The blocking of calcium channel with verapamil obviously increased the expression of CD34, CD45, and CD11b, thus indicating that the diminished calcium ion influx is coupled with the hematopoietic differentiation of MSCs under tumor conditions. Taken together, in a cancer environment, MSCs could effectively differentiate into immune hematopoietic cells, precisely macrophages. Diminished transient influx of Ca(2+) may mediate the hematopoietic differentiation of MSCs.