Mesenchymal Stem Cells In Scaffold Research Articles

Bone regeneration of mouse critical-sized calvarial defects with human mesenchymal stem cells in scaffold

Combination of tissue engineering and cell therapy represents a promising approach for bone regeneration. Human mesenchymal stem cells (hMSCs) have properties that include low immunogenicity, high proliferation rate, and multi-differentiation potential; therefore, they are an attractive seeding source for tissue engineering therapy. Here we found that hMSCs with a scaffold did not affect cell viability and osteogenic differentiation. We also investigated regenerative effect of hMSCs with the scaffold in a calvarial bone defect model. Formation of new bone was evaluated by micro-CT, histology and expression of osteogenic markers. The results clearly showed interesting evidence indicating that hMSCs with scaffold increased the formation of new bone and expression of osteogenic markers, compared to the empty and scaffold only groups. Overall, our results suggest that hMSCs with scaffold are suitable for stimulation of intense bone regeneration in critical-sized 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.

Can mesenchymal stem cell survive in hydroxyapatite sulphate?

Background: Many studies have reported the role of Mesenchymal Stem Cells (MSC) in treating fractures. In case with bone defect, fracture healing needs not only osteogenic but also osteoconductive component (scaffold). Hydroxyapatite calcium sulphate (HA-CaSO4) being widely used as bone void filler, may serve as scaffold for MSC. However, the effect of this scaffold to the viability of MSC has not been evaluated before.Methods: MSC were isolated from the iliac marrow of a Giant Flamish rabbit, and expanded in DMEM using histogradient density. After one week, they were sub-cultured in a 25cc TC flask (passage 1) and have the medium replaced every 3 days. During the subculture, we embedded a HA-CaSO4 pellet into the flask. The cells were evaluated under inverted microscope at a weekly interval.Results: At the first week, MSC are difficult to be identified in microscope due to the large number of HA-CaSO4 crystals. By the third week however MSC have grown and the HA-CaSO4 crystals can readily be washed off by medium replacement. By the fourth weeks, MSC can be still seen on microscope.Conclusion: HA-CaSO4 could serve as a good scaffold due to its pellet shape and easily absorbed, thus providing revascularization which is essential for bone healing.In addition, HA-CaSO4 does not interfere with MSC survival. (Med J Indones 2012;21:8-12)Keywords: Fracture healing, Hydroxyapatite Calcium Sulphate (HA-CaSO4), Mesenchymal Stem Cells (MSC)

Influence of different commercial scaffolds on the in vitro differentiation of human mesenchymal stem cells to nucleus pulposus-like cells

Cell-based therapies for regeneration of the degenerated intervertebral disc (IVD) are an alternative to current surgical intervention. Mesenchymal stem cells (MSCs), in combination with a scaffold, might be ideal candidates for regenerating nucleus pulposus (NP), the pressure-distributing part of the IVD. While the use of growth factors for MSCs differentiation currently receives major attention, in this study we compare the performance of sponge-like matrixes in supporting cell differentiation into NP-like cells. Four types matrixes approved as medical devices for other applications were tested as scaffolds for MSCs: two made of equine or porcine collagen, one of gelatin and one of chitosan. Bone marrow-derived human MSCs were seeded in these scaffolds or embedded in alginate, as a three-dimensional control. After five weeks in culture, NP-like differentiation of the cell-scaffold constructs was analyzed by qRT-PCR, histology, total DNA quantification, proteoglycan accumulation and immunohistochemistry. MSCs in collagen matrixes and gelatin produced more mRNA and proteins of the chondrogenic markers collagen type I, collagen type II (COL2) and aggrecan (ACAN), when compared with cells embedded in alginate or chitosan. Proteoglycan accumulation and cell survival were also higher in collagen and gelatin matrixes. Gene expression results were also confirmed by histological and immunohistochemical staining. In contrast to alginate control, the gene expression of the undesired bone marker osteopontin was lower in all tested groups. In porcine collagen supports, MSC expression ratio between COL2/ACAN closely resembled the expression of nucleus pulposus cells, but gene expression of recently described NP markers keratin19, PAX1 and FOXF1 was lower. Collagen supports provide a readily available, medically approved and effective scaffold for chondrogenic differentiation in vitro, but the phenotype of differentiated MSCs is not yet completely equivalent to that of NP cells.

Biocompatibility of absorbable gelatin sponge as bone mesenchymal stem cells scaffold

目的 探讨可吸收明胶海绵的生物相容性,为其作为骨髓间充质干细胞(BMSCs)的有效载体进行眼内移植奠定基础.方法 体外培养BMSCs,接种于可吸收明胶海绵,观察BMSCs的黏附、增生情况,采用乳鼠视网膜细胞条件分化液诱导,观察BMSCs的分化情况并行免疫组织化学鉴定.结果 在乳鼠视网膜条件分化液的环境中,接种后72 h,BMSCs伸出伪足黏附于可吸收明胶海绵表面.诱导后的细胞呈现神经元细胞的典型形态,免疫组织化学显示神经元特异性烯醇化酶(NSE)和巢蛋白(nestin)阳性.结论 BMSCs能够在可吸收明胶海绵上较好地黏附、增生及分化,可吸收明胶海绵具有良好的生物相容性。

In vivo and in vitro evaluation of an Acetobacter xylinum synthesized microbial cellulose membrane intended for guided tissue repair.

BackgroundBarrier materials as cellulose membranes are used for guided tissue repair. However, it is essential that the surrounding tissues accept the device. The present study histologically evaluated tissue reaction to a microbial cellulose membrane after subcutaneous implantation in mice. Furthermore, the interaction between mesenchymal stem cells and the biomaterial was studied in vitro to evaluate its ability to act as cellular scaffold for tissue engineering.MethodsTwenty-five Swiss Albino mice were used. A 10 × 10 mm cellulose membrane obtained through biosynthesis using Acetobacter xylinum bacteria was implanted into the lumbar subcutaneous tissue of each mouse. The mice were euthanatized at seven, 15, 30, 60, and 90 days, and the membrane and surrounding tissues were collected and examined by histology.ResultsA mild inflammatory response without foreign body reaction was observed until 30 days post-surgery around the implanted membrane. Polarized microscopy revealed that the membrane remained intact at all evaluation points. Scanning electron microscopy of the cellulose membrane surface showed absence of pores. The in vitro evaluation of the interaction between cells and biomaterial was performed through viability staining analysis of the cells over the biomaterial, which showed that 95% of the mesenchymal stem cells aggregating to the cellulose membrane were alive and that 5% were necrotic. Scanning electron microscopy showed mesenchymal stem cells with normal morphology and attached to the cellulose membrane surface.ConclusionThe microbial cellulose membrane evaluated was found to be nonresorbable, induced a mild inflammatory response and may prove useful as a scaffold for mesenchymal stem cells.

Chondrogenesis using mesenchymal stem cells and PCL scaffolds

We tested the in vitro feasibility of porous PCL (poly(epsilon-caprolactone)) as a scaffold for cartilage tissue engineering from mesenchymal stem cells (MSCs) and determined the effects of various surface treatments. Three porous PCL scaffold modifications were examined: (1) PCL/Pluronic F127, (2) PCL/collagen, and (3) PCL/Pluronic F127/collagen, in addition to (4) PCL-only. MSCs (5 x 10(5)) were seeded in PCL scaffolds of pore size 100-150 microm, and after 3 weeks of in vitro culture, MSC-scaffolds were investigated for gross appearance, DNA amount, glycosaminoglycan (GAG) content, chondrogenic gene expression, and histology. Grossly, the cell-scaffold complexes became harder, and were more easily manipulated with a forceps after 3 weeks of culture. The three surface-treated scaffolds had higher DNA contents than did the PCL-only scaffold, and the GAG contents in PCL/collagen and PCL/F127/collagen scaffolds were higher than those seen in the PCL-only scaffold. Real-time PCR showed that Sox-9 and COL2A1 mRNA levels were remarkably elevated in PCL/collagen and PCL/F127/collagen scaffolds versus the PCL-only scaffold. On the other hand, Col1A1 and Col10A1 mRNA levels were lower in the three modified PCL scaffolds than in the PCL-only scaffold. Histological findings generally concurred with GAG and RT-PCR findings, and demonstrated the affinity of PCL-based scaffolds for MSCs and the potentials of these scaffold to induce chondrogenic differentiation. Cells showed more differentiated appearance and more abundant extracellular matrix formation in PCL/collagen and PCL/collagen/F127 scaffolds. Our findings suggest that PCL-based porous scaffolds may be useful carriers for MSC transplantation in the cartilage tissue engineering field, and that collagen-based surface modifications further enhance the chondrogenic differentiation of MSCs.

Stability of Collagen Scaffold Implants for Animals with Iatrogenic Articular Cartilage Defects

Synthesis and characterization of biodegradable hydrogels based on collagen modified by addition of synthetic biodegradable copolymer intended for preparation of porous scaffolds for mesenchymal stem cells used for possible implantation to animals with articular surface defects was investigated. The synthetic biodegradable tri-block copolymer used was the block copolymer of polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA) (PEG-PLGA) endcapped with itaconic acid (ITA). The water-soluble carbodiimide and N-hydroxysuccimide system (EDC-NHS) was chosen as the cross-linking agent used to control the rate of hydrogel resorption. Dependence of the physical properties of the prepared hydrogels on the concentration of the EDC-NHS cross-linker, reaction time and concentration of PEG-PLGA-ITA copolymer was examined. Swelling behaviour, thermal stability, surface morphology and degradation rate were also characterized. Based on the obtained results, it can be concluded that increase in concentration of the cross-linking agent, as well as prolonged cross-linking time and increased amount of synthetic copolymer lead to enhanced thermal stability of the gels together with a reduced swelling ratio and degradation rate in saline. The resorption rate of these gels used in preparation of cartilage scaffolds can be controlled over a wide time interval by varying the collagen/(PEG-PLGA-ITA) blend composition or the conditions of the cross-linking reaction.

Human autologous plasma‐derived clot as a biological scaffold for mesenchymal stem cells in treatment of orthopedic healing

Recent advances in the isolation, expansion, and characterization of human mesenchymal stem cells (hMSCs) have raised the possibility of using them in cell therapies and tissue engineering for bone reconstruction. hMSCs, isolated from the bone marrow of eight normal adult patients, were minimally expanded ex vivo and pulsed twice toward osteogenic lineage. The cells were then included into autologous plasma-derived clots. Cytofluorimetric analysis, immunocytochemistry (osteopontin), histochemistry (alkaline phosphatase, Alcian blue, Von Kossa, and alizarin red staining), and viable/proliferation tests were performed to study both stem and differentiating cells. Although two short inductions increased osteogenic markers in hMSCs, inside the clot the cells were able to terminally differentiate into osteoblasts. Moreover, we show that the clot is able to sustain cell proliferation under appropriate cell culture conditions. Our results suggested that clot could be useful for hMSC delivery into the site of the lesion to promote bone formation. Moreover, the plasticity of this material allowed good in vitro hMSC spreading and proliferation. The advantages of using this autologous biological material are its biocompatibility and reabsorption; furthermore, using a gel as scaffold, it is possible to mold it to the shape of a bone cavity.

Combination of 3D tissue engineered scaffold and non-viral gene carrier enhance in vitro DNA expression of mesenchymal stem cells

The objective of this study is to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSC) by combination of 3-dimensional (3D) tissue engineered scaffolds and non-viral gene carrier. As a carrier of plasmid DNA, dextran-spermine cationic polysaccharide was prepared by means of reductive-amination between oxidized dextran and the natural oligoamine, spermine. As the MSC scaffold, collagen sponges reinforced by incorporation of poly(glycolic acid) (PGA) fibers were used. A complex of the cationized dextran and plasmid DNA of BMP-2 was impregnated into the scaffolds. MCS were seeded into each scaffold and cultured by a 3D culture method. When MSC were cultured in the PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by the cationized dextran-plasmid DNA complex impregnated into the scaffold than by the cationized dextran-plasmid DNA complex in 2-dimensional (2D) (tissue culture plate) culture method. The alkaline phosphatase activity and osteocalcin content of transfected MSC cultured in the PGA-reinforced sponge were significantly higher compared with 2D culture method. We conclude that combination of cationized dextran plasmid DNA complex and 3D tissue engineered scaffold was promising to promote the in vitro gene expression for MSC.

Impregnation of Plasmid DNA into Three-Dimensional Scaffolds and Medium Perfusion Enhancein VitroDNA Expression of Mesenchymal Stem Cells

This article describes the development of an in vitro culture system to enhance the expression of a plasmid DNA for mesenchymal stem cells (MSCs) by a combination of plasmid DNA impregnation into three-dimensional cell scaffolds and culture methods. Gelatin was cationized by introducing spermine to the carboxyl groups for complexation with the plasmid DNA. As the MSC scaffold, poly(glycolic acid) (PGA) fiber fabrics, collagen sponges, and collagen sponges reinforced by incorporation of PGA fibers were used. A complex of cationized gelatin and plasmid DNA encoding bone morphogenetic protein 2 (BMP-2) was impregnated into the scaffolds. Plasmid DNA was released from PGA-reinforced collagen sponge for longer than from the other scaffolds. MCS were seeded into each type of scaffold and cultured by static, stirring, and perfusion methods. When MSCs were cultured in PGA-reinforced sponge, the level of BMP-2 expression was significantly enhanced by perfusion culture compared with the other culture methods, and the time of expression was prolonged. Irrespective of the culture method, the expression level was significantly higher from plasmid DNA impregnated in scaffold than by plasmid DNA in medium. The alkaline phosphatase activity and osteocalcin content of MSCs cultured in PGA-reinforced sponge by the perfusion method were significantly higher compared with those of other methods, and a significantly higher amount of plasmid DNA internalized into MSCs was observed. We conclude that a combination of plasmid DNA-impregnated PGA-reinforced sponge and the perfusion method was promising to promote in vitro gene expression for MSCs.

Type I collagen combined with a recombinant TGF-beta serves as a scaffold for mesenchymal stem cells.

Type I collagen combined with a recombinant TGF-beta serves as a scaffold for mesenchymal stem cells.