Scaffolds for engineering smooth muscle under cyclic mechanical strain conditions

Cyclic mechanical strain has been demonstrated to enhance the development and function of engineered smooth muscle (SM) tissues, but appropriate scaffolds for engineering tissues under conditions of cyclic strain are currently lacking. These scaffolds must display elastic behavior, and be capable of inducing an appropriate smooth muscle cell (SMC) phenotype in response to mechanical signals. In this study, we have characterized several scaffold types commonly utilized in tissue engineering applications in order to select scaffolds that exhibit elastic properties under appropriate cyclic strain conditions. The ability of the scaffolds to promote an appropriate SMC phenotype in engineered SM tissues under cyclic strain conditions was subsequently analyzed. Poly(L-lactic acid)-bonded polyglycolide fiber-based scaffolds and type I collagen sponges exhibited partially elastic mechanical properties under cyclic strain conditions, although the synthetic polymer scaffolds demonstrated significant permanent deformation after extended times of cyclic strain application. SM tissues engineered with type I collagen sponges subjected to cyclic strain were found to contain more elastin than control tissues, and the SMCs in these tissues exhibited a contractile phenotype. In contrast, SMCs in control tissues exhibited a structure more consistent with the nondifferentiated, synthetic phenotype. These studies indicate the appropriate choice of a scaffold for engineering tissues in a mechanically dynamic environment is dependent on the time frame of the mechanical stimulation, and elastic scaffolds allow for mechanically directed control of cell phenotype in engineered tissues.     

Cyclic mechanical strain regulates the development of engineered smooth muscle tissue.

We show that the appropriate combinations of mechanical stimuli and polymeric scaffolds can enhance the mechanical properties of engineered tissues. The mechanical properties of tissues engineered from cells and polymer scaffolds are significantly lower than the native tissues they replace. We hypothesized that application of mechanical stimuli to engineered tissues would alter their mechanical properties. Smooth muscle tissue was engineered on two different polymeric scaffolds and subjected to cyclic mechanical strain. Short-term application of strain increased proliferation of smooth muscle cells (SMCs) and expression of collagen and elastin, but only when SMCs were adherent to specific scaffolds. Long-term application of cyclic strain upregulated elastin and collagen gene expression and led to increased organization in tissues. This resulted in more than an order of magnitude increase in the mechanical properties of the tissues.     

Self-assembly of elastin-based peptides into the ECM: the importance of integrins and the elastin binding protein in elastic fiber assembly

The formation of a suitable extracellular matrix (ECM) that promotes cell adhesion, organization, and proliferation is essential within biomaterial scaffolds for tissue engineering applications. In this work, short elastin mimetic peptide sequences, EM-19 and EM-23, were engineered to mimic the active motifs of human elastin in hopes that they can stimulate ECM development in synthetic polymer scaffolds. Each peptide was incubated with human aortic smooth muscle cells (SMCs) and elastin and desmosine production were quantified after 48 h. EM-19 inhibited elastin production through competitive binding phenomena with the elastin binding protein (EBP), whereas EM-23, which contains an RGDS domain, induces recovery of elastin production at higher concentrations, alluding to a higher binding affinity for the integrins than for the EBP and the involvement of integrins in elastin production. Colocalization of each peptide with the elastin matrix was confirmed using immunofluorescent techniques. Our data suggest that with appropriate cell-binding motifs, we can simulate the cross-linking of tropoelastin into the developing elastin matrix using short peptide sequences. The potential for increased cell adhesion and the incorporation of elastin chains into tissue engineering scaffolds make these peptides attractive bioactive moieties that can easily be incorporated into synthetic biomaterials to induce ECM formation.     

A mechanical composite spheres analysis of engineered cartilage dynamics

In the preparation of bioengineered reparative strategies for damaged or diseased tissues, the processes of biomaterial degradation and neotissue synthesis combine to affect the developing mechanical state of multiphase, composite engineered tissues. Here, cell-polymer constructs for engineered cartilage have been fabricated by seeding chondrocytes within three-dimensional scaffolds of biodegradable polymers. During culture, synthetic scaffolds degraded passively as the cells assembled an extracellular matrix (ECM) composed primarily of glycosaminoglycan and collagen. Biochemical and biomechanical assessment of the composite (cells, ECM, and polymer scaffold) were modeled at a unit-cell level to mathematically solve stress-strain relationships and thus construct elastic properties (n=4 samples per seven time points). This approach employed a composite spheres, micromechanical analysis to determine bulk moduli of: (1) the cellular-ECM inclusion within the supporting scaffold structure; and (2) the cellular inclusion within its ECM. Results indicate a dependence of constituent volume fractions with culture time (p<0.05). Overall mean bulk moduli were variably influenced by culture, as noted for the cell-ECM inclusion (K(c-m)=29.7 kPa, p=0.1439), the cellular inclusion (K(c)=5.5 kPa, p=0.0067), and its surrounding ECM (K(m)=373.9 kPa, p=0.0748), as well as the overall engineered construct (K=165.0 kPa, p=0.6899). This analytical technique provides a framework to describe the time-dependent contribution of cells, accumulating ECM, and a degrading scaffold affecting bioengineered construct mechanical properties.     

Strategies for enhancing the accumulation and retention of extracellular matrix in tissue-engineered cartilage cultured in bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min(-1)) and gradually increasing (0.075-0.2 mL min(-1)) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0-4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8-5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min(-1). GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.     

Enhanced cartilage tissue engineering by sequential exposure of chondrocytes to FGF-2 during 2D expansion and BMP-2 during 3D cultivation

Bovine calf articular chondrocytes, either primary or expanded in monolayers (2D) with or without 5 ng/ml fibroblast growth factor-2 (FGF-2), were cultured on three-dimensional (3D) biodegradable polyglycolic acid (PGA) scaffolds with or without 10 ng/ml bone morphogenetic protein-2 (BMP-2). Chondrocytes expanded without FGF-2 exhibited high intensity immunostaining for smooth muscle alpha-actin (SMA) and collagen type I and induced shrinkage of the PGA scaffold, thus resembling contractile fibroblasts. Chondrocytes expanded in the presence of FGF-2 and cultured 6 weeks on PGA scaffolds yielded engineered cartilage with 3.7-fold higher cell number, 4.2-fold higher wet weight, and 2.8-fold higher wet weight glycosaminoglycan (GAG) fraction than chondrocytes expanded without FGF-2. Chondrocytes expanded with FGF-2 and cultured on PGA scaffolds in the presence of BMP-2 for 6 weeks yielded engineered cartilage with similar cellularity and size, 1.5-fold higher wet weight GAG fraction, and more homogenous GAG distribution than the corresponding engineered cartilage cultured without BMP-2. The presence of BMP-2 during 3D culture had no apparent effect on primary chondrocytes or those expanded without FGF-2. In summary, the presence of FGF-2 during 2D expansion reduced chondrocyte expression of fibroblastic molecules and induced responsiveness to BMP-2 during 3D cultivation on PGA scaffolds.     

Optimizing seeding and culture methods to engineer smooth muscle tissue on biodegradable polymer matrices

The engineering of functional smooth muscle (SM) tissue is critical if one hopes to successfully replace the large number of tissues containing an SM component with engineered equivalents. This study reports on the effects of SM cell (SMC) seeding and culture conditions on the cellularity and composition of SM tissues engineered using biodegradable matrices (5 x 5 mm, 2-mm thick) of polyglycolic acid (PGA) fibers. Cells were seeded by injecting a cell suspension into polymer matrices in tissue culture dishes (static seeding), by stirring polymer matrices and a cell suspension in spinner flasks (stirred seeding), or by agitating polymer matrices and a cell suspension in tubes with an orbital shaker (agitated seeding). The density of SMCs adherent to these matrices was a function of cell concentration in the seeding solution, but under all conditions a larger number (approximately 1 order of magnitude) and more uniform distribution of SMCs adherent to the matrices were obtained with dynamic versus static seeding methods. The dynamic seeding methods, as compared to the static method, also ultimately resulted in new tissues that had a higher cellularity, more uniform cell distribution, and greater elastin deposition. The effects of culture conditions were next studied by culturing cell-polymer constructs in a stirred bioreactor versus static culture conditions. The stirred culture of SMC-seeded polymer matrices resulted in tissues with a cell density of 6.4 +/- 0.8 x 10(8) cells/cm3 after 5 weeks, compared to 2.0 +/- 1.1 x 10(8) cells/cm3 with static culture. The elastin and collagen synthesis rates and deposition within the engineered tissues were also increased by culture in the bioreactors. The elastin content after 5-week culture in the stirred bioreactor was 24 +/- 3%, and both the elastin content and the cellularity of these tissues are comparable to those of native SM tissue. New tissues were also created in vivo when dynamically seeded polymer matrices were implanted in rats for various times. In summary, the system defined by these studies shows promise for engineering a tissue comparable in many respects to native SM. This engineered tissue may find clinical applications and provide a tool to study molecular mechanisms in vascular development.     

Specific interactions between Porphyromonas gingivalis fimbriae and human extracellular matrix proteins

The interactions of the extracellular matrix (ECM) proteins (laminin, elastin, fibronectin, type I collagen, thrombospondin and vitronectin) with the fimbriae of Porphyromonas gingivalis were analyzed based on surface plasmon resonance (SPR) spectroscopy using a biomolecular interaction analyzing system (BIAcore). The BIAcore profiles demonstrated that fimbriae specifically bound to all of the ECM proteins with significant association constants (Ka). Vitronectin showed the highest affinity to fimbriae (Ka = 3.79 x 10(6) M-1), while the affinity of laminin was lowest (Ka = 2.15 x 10(6) M-1). A synthetic peptide which is a potent inhibitor of fimbrial binding to salivary proteins was not significantly effective on the fimbrial interactions with the ECM proteins. Using polystyrene microtiter plates revealed that P. gingivalis fimbriae bound markedly to immobilized fibronectin and type I collagen, while the interaction of fimbriae with the other ECM proteins was not clearly demonstrated. These results suggest that interactions between fimbriae and the ECM proteins occur with specific affinities which are not mediated by mechanisms identical to those of salivary proteins. It was also shown that SPR spectroscopy is a useful method to analyze these specific interactions.     

Extracellular matrix protein mediated regulation of the osteoblast differentiation of bone marrow derived human mesenchymal stem cells

The biomimetic approach of tissue engineering exploits the favorable properties of the extracellular matrix (ECM), to achieve better scaffold performance and tissue regeneration. ECM proteins regulate cell adhesion and differentiation through integrin mediated signal transduction. In the present study, we have examined the role of ECM proteins such as collagen type I, fibronectin, laminin and vitronectin in regulating the proliferation and osteogenic differentiation of bone marrow derived human mesenchymal stem cells (hMSCs). hMSCs were grown on selected ECM protein treated tissue culture plates. The growth kinetics was assessed by calculating the doubling time of the cells on different ECM treated plates. The cells were directed to osteoblast lineage by growing them in osteogenic induction media for 21 day. Differentiation was evaluated at different time points by osteoblast differentiation associated gene expression, alkaline phosphatase (ALP) activity, histochemical staining for mineralized matrix and calcium quantification. The doubling time of hMSCs cultured on collagen type I was significantly low, which was followed by laminin and fibronectin treated plates. However, doubling time of hMSCs cultured on vitronectin treated plate was not significantly different than that of the untreated control. High ALP gene (ALPL) expression and associated enhancement of mineralization were observed on collagen type I, fibronectin and vitronectin treated plates. Collagen type I showed early onset of mineralization with high ALP activity and up-regulation of osteopontin, ALPL, bone sialoprotein and osteocalcin genes. Vitronectin also up-regulated these genes and showed the highest amount of calcium in the secreted mineral matrix. Therefore, we conclude that, ECM proteins indeed modified the growth patterns and induced the osteoblast differentiation of hMSCs. Our findings have significant implication for bone tissue engineering applications.     

A structural model for the flexural mechanics of nonwoven tissue engineering scaffolds

The development of methods to predict the strength and stiffness of biomaterials used in tissue engineering is critical for load-bearing applications in which the essential functional requirements are primarily mechanical. We previously quantified changes in the effective stiffness (E) of needled nonwoven polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) scaffolds due to tissue formation and scaffold degradation under three-point bending. Toward predicting these changes, we present a structural model for E of a needled nonwoven scaffold in flexure. The model accounted for the number and orientation of fibers within a representative volume element of the scaffold demarcated by the needling process. The spring-like effective stiffness of the curved fibers was calculated using the sinusoidal fiber shapes. Structural and mechanical properties of PGA and PLLA fibers and PGA, PLLA, and 50:50 PGA/PLLA scaffolds were measured and compared with model predictions. To verify the general predictive capability, the predicted dependence of E on fiber diameter was compared with experimental measurements. Needled nonwoven scaffolds were found to exhibit distinct preferred (PD) and cross-preferred (XD) fiber directions, with an E ratio (PD/XD) of approximately 3:1. The good agreement between the predicted and experimental dependence of E on fiber diameter (R2 = 0.987) suggests that the structural model can be used to design scaffolds with E values more similar to native soft tissues. A comparison with previous results for cell-seeded scaffolds (Engelmayr, G. C., Jr., et al., 2005, Biomaterials, 26(2), pp. 175-187) suggests, for the first time, that the primary mechanical effect of collagen deposition is an increase in the number of fiber-fiber bond points yielding effectively stiffer scaffold fibers. This finding indicated that the effects of tissue deposition on needled nonwoven scaffold mechanics do not follow a rule-of-mixtures behavior. These important results underscore the need for structural approaches in modeling the effects of engineered tissue formation on nonwoven scaffolds, and their potential utility in scaffold design.     

Composite scaffolds for the engineering of hollow organs and tissues

Several types of synthetic and naturally derived biomaterials have been used for augmenting hollow organs and tissues. However, each has desirable traits which were exclusive of the other. We fabricated a composite scaffold and tested its potential for the engineering of hollow organs in a bladder tissue model. The composite scaffolds were configured to accommodate a large number of cells on one side and were designed to serve as a barrier on the opposite side. The scaffolds were fabricated by bonding a collagen matrix to PGA polymers with threaded collagen fiber stitches. Urothelial and bladder smooth muscle cells were seeded on the composite scaffolds, and implanted in mice for up to 4 weeks and analyzed. Both cell types readily attached and proliferated on the scaffolds and formed bladder tissue-like structures in vivo. These structures consisted of a luminal urothelial layer, a collagen rich compartment and a peripheral smooth muscle layer. Biomechanical studies demonstrated that the tissues were readily elastic while maintaining their pre-configured structures. This study demonstrates that a composite scaffold can be fabricated with two completely different polymer systems for the engineering of hollow organs. The composite scaffolds are biocompatible, possess adequate physical and structural characteristics for bladder tissue engineering, and are able to form tissues in vivo. This scaffold system may be useful in patients requiring hollow organ replacement.     

Collagen scaffolds for tissue engineering

There are two major approaches to tissue engineering for regeneration of tissues and organs. One involves cell-free materials and/or factors and one involves delivering cells to contribute to the regeneraion process. Of the many scaffold materials being investigated, collagen type I, with selective removal of its telopeptides, has been shown to have many advantageous features for both of these approaches. Highly porous collagen lattice sponges have been used to support in vitro growth of many types of tissues. Use of bioreactors to control in vitro perfusion of medium and to apply hydrostatic fluid pressure has been shown to enhance histogenesis in collagen scaffolds. Collagen sponges have also been developed to contain differentiating-inducing materials like demineralized bone to stimulate differentiation of cartilage tissue both in vitro and in vivo.     

接种方式对细胞在三维材料中接种率和分布的影响

考察了静态和动态接种方式对成纤维细胞在胶原壳聚糖支架材料中接种率和分布的影响。将人成纤维细胞制成细胞悬液,分别采用静态接种、转瓶接种和灌注接种方式将细胞接入三维胶原壳聚糖海绵。通过MTT法和切片HE染色分别考察细胞接种率及细胞在三维材料中的分布。实验结果表明：在低的接种密度下静态接种有较高的接种率(889%),但随着接种密度的增加接种率下降显著,细胞结团且分布不均匀;转瓶接种的接种率约为60%,细胞分布也不均匀;灌注接种的接种率始终维持在77%以上,能得到高的起始细胞密度,且细胞分布均匀,是一种理想的接种方式。细胞接种方式的优化为改善工程化组织的结构和功能、缩短体外构建时间奠定了基础。     

Fabrication and evaluation of biomimetic-synthetic nanofibrous composites for soft tissue regeneration

Electrospun scaffolds hold promise for the regeneration of dense connective tissues, given their nanoscale topographies, provision of directional cues for infiltrating cells and versatile composition. Synthetic slow-degrading scaffolds provide long-term mechanical support and nanoscale instructional cues; however, these scaffolds suffer from a poor infiltration rate. Alternatively, nanofibrous constructs formed from natural biomimetic materials (such as collagen) rapidly infiltrate but provide little mechanical support. To take advantage of the positive features of these constructs, we have developed a composite scaffold consisting in both a biomimetic fiber fraction (i.e., Type I collagen nanofibers) together with a traditional synthetic (i.e., poly-[ε-caprolactone], PCL) fiber fraction. We hypothesize that inclusion of biomimetic elements will improve initial cell adhesion and eventual scaffold infiltration, whereas the synthetic elements will provide controlled and long-term mechanical support. We have developed a method of forming and crosslinking collagen nanofibers by using the natural crosslinking agent genipin (GP). Further, we have formed composites from collagen and PCL and evaluated the long-term performance of these scaffolds when seeded with mesenchymal stem cells. Our results demonstrate that GP crosslinking is cytocompatible and generates stable nanofibrous type I collagen constructs. Composites with varying fractions of the biomimetic and synthetic fiber families are formed and retain their collagen fiber fractions during in vitro culture. However, at the maximum collagen fiber fractions (20%), cell ingress is limited compared with pure PCL scaffolds. These results provide a new foundation for the development and optimization of biomimetic/synthetic nanofibrous composites for in vivo tissue engineering.     

Tissue engineering of human cartilage in bioreactors using single and composite cell-seeded scaffolds

Chondrocytes isolated from human fetal epiphyseal cartilage were seeded under mixed conditions into 15-mm-diameter polyglycolic acid (PGA) scaffolds and cultured in recirculation column bioreactors to generate cartilage constructs. After seeding, the cell distributions in thick (4.75 mm) and thin (2.15 mm) PGA disks were nonuniform, with higher cell densities accumulating near the top surfaces. Composite scaffolds were developed by suturing together two thin PGA disks after seeding to manipulate the initial cell distribution before bioreactor culture. The effect of medium flow direction in the bioreactors, including periodic reversal of medium flow, was also investigated. The quality of the tissue-engineered cartilage was assessed after 5 weeks of culture in terms of the tissue wet weight, glycosaminoglycan (GAG), total collagen and collagen type II contents, histological analysis of cell, GAG and collagen distributions, and immunohistochemical analysis of collagen types I and II. Significant enhancement in construct quality was achieved using composite scaffolds compared with single PGA disks. Operation of the bioreactors with periodic medium flow reversal instead of unidirectional flow yielded further improvements in tissue weight and GAG and collagen contents with the composite scaffolds. At harvest, the constructs contained GAG concentrations similar to those measured in ex vivo human adult articular cartilage; however, total collagen and collagen type II levels were substantially lower than those in adult tissue. This study demonstrates that the location of regions of high cell density in the scaffold coupled with application of dynamic bioreactor operating conditions has a significant influence on the quality of tissue-engineered cartilage.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.     

Chondrogenic differentiation of human bone marrow-derived mesenchymal stromal cells in a three-dimensional environment

Cell therapy combined with biomaterial scaffolds is used to treat cartilage defects. We hypothesized that chondrogenic differentiation bone marrow-derived mesenchymal stem cells (BM-MSCs) in three-dimensional biomaterial scaffolds would initiate cartilaginous matrix deposition and prepare the construct for cartilage regeneration in situ. The chondrogenic capability of human BM-MSCs was first verified in a pellet culture. The BM-MSCs were then either seeded onto a composite scaffold rhCo-PLA combining polylactide and collagen type II (C2) or type III (C3), or commercial collagen type I/III membrane (CG). The BM-MSCs were either cultured in a proliferation medium or chondrogenic culture medium. Adult human chondrocytes (ACs) served as controls. After 3, 14, and 28 days, the constructs were analyzed with quantitative polymerase chain reaction and confocal microscopy and sulfated glycosaminoglycans (GAGs) were measured. The differentiated BM-MSCs entered a hypertrophic state by Day 14 of culture. The ACs showed dedifferentiation with no expression of chondrogenic genes and low amount of GAG. The CG membrane induced the highest expression levels of hypertrophic genes. The two different collagen types in composite scaffolds yielded similar results. Regardless of the biomaterial scaffold, culturing BM-MSCs in chondrogenic differentiation medium resulted in chondrocyte hypertrophy. Thus, caution for cell fate is required when designing cell-biomaterial constructs for cartilage regeneration.     

Evaluating osteochondral defect repair potential of autologous rabbit bone marrow cells on type II collagen scaffold

The feasibility of using genipin cross-linked type II collagen scaffold with rabbit bone marrow mesenchymal stem cells (RBMSCs) to repair cartilage defect was herein studied. Induction of RBMSCs into chondrocytic phenotype on type II collagen scaffold in vitro was conducted using TGF-β 3 containing medium. After 3-weeks of induction, chondrocytic behavior, including marker genes expression and specific extracellular matrix (ECM) secretion, was observed. In the in vivo evaluation experiment, the scaffolds containing RBMSCs without prior induction were autologous implanted into the articular cartilage defects made by subchondral drilling. The repairing ability was evaluated. After 2 months, chondrocyte-like cells with lacuna structure and corresponding ECM were found in the repaired sites without apparent inflammation. After 24 weeks, we could easily find cartilage structure the same with normal cartilage in the repair site. In conclusion, it was shown that the scaffolds in combination of in vivo conditions can induce RBMSCs into chondrocytes in repaired area and would be a possible method for articular cartilage repair in clinic and cartilage tissue engineering.     

Osteogenic differentiation of miniature pig mesenchymal stem cells in 2D and 3D environment

Mesenchymal stem cells (MSCs) have been repeatedly shown to be able to repair bone defects. The aim of this study was to characterize the osteogenic differentiation of miniature pig MSCs and markers of this differentiation in vitro. Flow-cytometrically characterized MSCs were seeded on cultivation plastic (collagen I and vitronectin coated/uncoated) or plasma clot (PC)/plasma-alginate clot (PAC) scaffolds and differentiated in osteogenic medium. During three weeks of differentiation, the formation of nodules and deposition of calcium were visualized by Alizarin Red Staining. In addition, the production of alkaline phosphatase (ALP) activity was quantitatively detected by fluorescence. The expression of osteopontin, osteonectin and osteocalcin were assayed by immunohistochemistry and Western Blot analysis. We revealed a decrease of osteopontin expression in 2D and 3D environment during differentiation. The weak initial osteonectin signal, culminating on 7(th) or 14(th) day of differentiation, depends on collagen I and vitronectin coating in 2D system. The highest activity of ALP was detected on 21(th) day of osteogenic differentiation. The PC scaffolds provided better conditions for osteogenic differentiation of MSCs than PAC scaffolds in vitro. We also observed expected effects of collagen I and vitronectin on the acceleration of osteogenic differentiation of miniature pig MSC. Our results indicate similar ability of miniature pig MSCs osteogenic differentiation in 2D and 3D environment, but the expression of osteogenic markers in scaffolds and ECM coated monolayers started earlier than in the monolayers without ECM.     

Galectin 1 inhibits incorporation of vitronectin and chondroitin sulfate B into the extracellular matrix of human vascular smooth muscle cells

Galectin-1, a beta-galactoside-binding dimeric lectin, interacts with the extracellular matrix (ECM) of smooth muscle cells (SMCs) and with particular ECM proteins. Enrichment of the ECM with galectin-1 affects adhesion and proliferation of cultured SMCs. Here we investigated whether galectin-1 (1) interacts with glycosaminoglycan (GAG) chains, (2) cross-links between ligands and facilitates the incorporation of GAGs, vitronectin and plasma fibronectin in the ECM of vascular SMCs. A recombinant galectin-1 fusion protein GalH, used in this study, formed dimers and interacted with ECM proteins. GAG chains inhibited these interactions. Among the studied GAG chains, only chondroitin sulfate B interacted with GalH in beta-galactoside-dependent manner. GalH did not bridge between ECM proteins on solid phase and [125I]-labelled ECM proteins or GAGs in solution. The ECM incorporated less vitronectin in the presence of soluble GalH. GalH-enriched ECM incorporated less vitronectin and chondroitin sulfate B. The ECM partially depleted of endogenous galectins incorporated more chondroitin sulfate B compared to untreated ECM. These results suggest that galectin-1 is likely to be involved in the ECM assembly affecting incorporation of some ECM components important for SMC behaviour.