In vitro characterization of an artificial dermal scaffold

The treatment of extensive burn injuries has been enhanced by the development of artificial skin substitutes. Integra Artificial Skin, an acellular collagen-glycosaminoglycan (C-GAG) dermal equivalent requires a two-stage grafting procedure. However, preseeding the C-GAG dermal equivalent with cultured fibroblasts and keratinocytes, with the aim of performing a single-stage grafting procedure, may be beneficial in terms of replacing the requirement for traditional split-skin grafts. In this comparative in vitro study, the interactions of cultured human dermal fibroblasts and epidermal keratinocytes in Integra Artificial Skin in comparison to cadaver deepidermalized dermis (DED) was investigated. An increase in cell proliferation and migration in the C-GAG dermal equivalent was observed over time. Cocultures of fibroblasts and keratinocytes on both dermal equivalents showed positive expression of proliferation, differentiation, and extracellular matrix (ECM) protein markers. Organization of keratinocytes in the epidermal layers of DED composites were better compared to the C-GAG composites. Deposition of ECM proteins was enhanced in the presence of keratinocytes in both dermal equivalents. Results demonstrate that in vitro the C-GAG dermal equivalent is biocompatible for cell attachment, migration, proliferation, and differentiation. Preseeding Integra Artificial Skin with cultured autologous fibroblasts and keratinocytes for in vivo application, as a single-stage grafting procedure, warrants testing. A better clinical outcome may be achieved as shown by our in vitro results of the coculture composites.     

A cartilage ECM-derived 3-D porous acellular matrix scaffold for in vivo cartilage tissue engineering with PKH26-labeled chondrogenic bone marrow-derived mesenchymal stem cells

We developed a natural, acellular, 3-D interconnected porous scaffold derived from cartilage extracellular matrix (ECM). Human cartilage was physically shattered, then decellularized sequentially with use of hypotonic buffer, TritonX-100, and a nuclease solution and made into a suspension. The scaffold was fabricated by simple freeze-drying and cross-linking techniques. On histology, scaffolds showed most of the ECM components after removal of the cell fragments, and scanning electron microscopy revealed a 3-D interconnected porous structure. Cellular viability assay revealed no cytotoxic effects. In vitro study showed that the novel scaffold could provide a suitable 3-D environment to support the adheration, proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) to chondrocytes in culture with chondrogenic medium after 21 days. Chondrogenically induced BMSCs labeled with fluorescent dye PKH26 were then grown on scaffolds and implanted subcutaneously into nude mice. Four weeks later, cartilage-like tissue formed, with positive staining for Safranin O, tuoluidine blue and collagen II. Cells in the samples seemed to confirm that they originated from the labeled BMSCs, as confirmed by in vivo fluorescent imaging and immunofluorescence examination. In conclusion, the cartilage ECM-derived porous scaffold shows potential as biomaterial for cartilage tissue engineering, and PKH26 fluorescent labeling and in vivo fluorescent imaging can be useful for cell tracking and analyzing cell-scaffold constructs in vivo.     

Neural tissue engineering and biohybridized microsystems for neurobiological investigation in vitro (Part 1)

Advances in neural tissue engineering have resulted in the development and implementation of three-dimensional (3-D) neural cellular constructs, which may serve as neurofidelic in vitro investigational platforms. In addition, interfacing these 3-D cellular constructs with micro-fluidic and/or micro-electrical systems has created biohybridized platforms, providing unprecedented 3-D microenvironmental control and allowing noninvasive probing and manipulation of cultured neural cells. Cells in the brain interact within a complex, multicellular environment with tightly coupled 3-D cell-cell/cell-extracellular matrix (ECM) interactions; yet most in vitro models utilize planar systems lacking in vivo-like ECM. As such, neural cultures with cells distributed throughout a thick (> 500 microm), bioactive extracellular matrix may provide a more physiologically relevant setting to study neurobiological phenomena than traditional planar cultures. This review presents an overview of 2-D versus 3-D culture models and the state of the art in 3-D neural cell-culture systems. We then detail our efforts to engineer a range of 3-D neural cellular constructs by systematically varying parameters such as cell composition, cell density, matrix constituents, and mass transport. The ramifications on neural cell survival, function, and network formation based on these parameters are specifically addressed. These 3-D neural cellular constructs may serve as powerful investigational platforms for the study of basic neurobiology, network neurophysiology, injury/disease mechanisms, pharmacological screening, or test-beds for cell replacement therapies. Furthermore, while survival and growth of neural cells within 3-D constructs poses many challenges, optimizing in vitro constructs prior to in vivo implementation offers a sound bioengineering design approach.     

In vitro cartilage tissue engineering with 3D porous aqueous-derived silk scaffolds and mesenchymal stem cells

Adult cartilage tissue has limited self-repair capacity, especially in the case of severe damages caused by developmental abnormalities, trauma, or aging-related degeneration like osteoarthritis. Adult mesenchymal stem cells (MSCs) have the potential to differentiate into cells of different lineages including bone, cartilage, and fat. In vitro cartilage tissue engineering using autologous MSCs and three-dimensional (3-D) porous scaffolds has the potential for the successful repair of severe cartilage damage. Ideally, scaffolds designed for cartilage tissue engineering should have optimal structural and mechanical properties, excellent biocompatibility, controlled degradation rate, and good handling characteristics. In the present work, a novel, highly porous silk scaffold was developed by an aqueous process according to these criteria and subsequently combined with MSCs for in vitro cartilage tissue engineering. Chondrogenesis of MSCs in the silk scaffold was evident by real-time RT-PCR analysis for cartilage-specific ECM gene markers, histological and immunohistochemical evaluations of cartilage-specific ECM components. Dexamethasone and TGF-beta3 were essential for the survival, proliferation and chondrogenesis of MSCs in the silk scaffolds. The attachment, proliferation, and differentiation of MSCs in the silk scaffold showed unique characteristics. After 3 weeks of cultivation, the spatial cell arrangement and the collagen type-II distribution in the MSCs-silk scaffold constructs resembles those in native articular cartilage tissue, suggesting promise for these novel 3-D degradable silk-based scaffolds in MSC-based cartilage repair. Further in vivo evaluation is necessary to fully recognize the clinical relevance of these observations.     

Engineered dermal equivalent tissue in vitro by assembly of microtissue precursors

Tissue-engineered constructs can be fabricated by the assembly of smaller building blocks in order to mimic much of the native biology that is often made from repeating functional units. Our aim was to realize a three-dimensional (3-D) tissue-like construct in vitro by inducing the assembly of functional micrometric tissue precursors (μTPs). μTPs were obtained by dynamic cell seeding of bovine fibroblasts on porous gelatine microcarriers using a spinner flask bioreactor. During the dynamic seeding, cells adhered, proliferated and synthesized a thin layer of extracellular matrix (ECM) in and around the macroporous beads, generating the μTPs. The analysis showed that the ECM produced was rich in type I collagen. The cells and ECM layer around the μTPs allowed their biological sintering via cell–cell and cell–matrix interaction after only a few days of dynamic seeding. The assembling ability of μTPs was exploited by placing them in a maturation chamber. After 1 week of culture disc-shaped constructs (1 cm in diameter, 1 mm in thickness) of completely assembled μTPs were collected. The biohybrid obtained presented both a homogeneous and compact aspect. Moreover, histological and immunohistochemical analyses revealed an abundant ECM, rich in type I collagen, interconnecting the μTPs. The results obtained in this survey pave the way to realizing a 3-D dermal tissue equivalent by means of a bottom-up tissue engineering approach.     

Gelatine methacrylamide-based hydrogels: An alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5–7% w/v) strongly influenced hydrogel stiffness (0.5 ± 0.2 kPa to 9.0 ± 1.8 kPa) but had little effect on solute diffusion. The diffusion coefficient of 70 kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4 kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33–37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.     

Disulfide-crosslinked hyaluronan-gelatin sponge: growth of fibrous tissue in vivo

The modification of hyaluronan (HA) and gelatin using dithiobis(propanoic dihydrazide) (DTP) has provided two thiolated macromolecular components of the extracellular matrix (ECM), specifically HA-DTPH and gelatin-DTPH. Blends of these thiolated ECM components were crosslinked in air to form hydrogels that were interpenetrating disulfide-crosslinked networks. Lyophilization of the hydrogels afforded sponge-like macroporous scaffolds suitable for cell attachment and proliferation. Increasing percentages of gelatin-DTPH (0, 25, 50, and 75%) were blended with HA-DTPH, and the resulting sponges were evaluated in vitro and in vivo as scaffolds for tissue engineering by seeding with human tracheal scar (HTS) fibroblasts. While cells failed to attach and grow in HA-only sponges, the gelatin-modified HA sponges promoted cell adhesion, proliferation, and spreading in vitro. Optimal attachment and growth was observed with 50% gelatin-HA sponges. Cell attachment to the gelatin-HA sponge could be blocked by preincubation of cells with a soluble fibronectin peptide Gly-Arg-Gly-Asp (GRGD). Finally, HTS fibroblast-seeded gelatin-HA sponges were implanted into the flanks of nude mice and evaluated at 2 and 8 weeks postimplantation. The sponges were fully biocompatible and new fibrous tissue formed, gradually replacing the sponge-like scaffold. The gelatin-HA sponges act as synthetic, macroporous, covalent mimics of the ECM and constitute novel scaffolds for cell growth and tissue augmentation.     

The performance of human dental pulp stem cells on different three-dimensional scaffold materials

The aim of this study was to investigate the in vitro and in vivo behavior of human dental pulp stem cells (DPSCs) isolated from impacted third molars, when seeded onto different 3-dimensional (3-D) scaffold materials: i.e. a spongeous collagen, a porous ceramic, and a fibrous titanium mesh. Scaffolds were loaded with DPSC, and subsequently divided into two groups. The first group was cultured in osteogenic differentiation medium in vitro for 4 weeks. The second group of samples was implanted subcutaneously in nude mice for 6 or 12 weeks. Samples cultured in vitro were analyzed by scanning electron microscopy and RT-PCR for dentin sialophosphoprotein (DSPP) expression. In vivo samples were evaluated by histology, RT-PCR and immunohistochemistry. The results indicated that in vitro, cells developed abundant deposition of mineralized extracellular matrix (ECM) with expression of DSPP in all 3-D materials. The simultaneous implantation experiment showed formation of tissue that was DSPP positive in all three scaffolds materials. However, the aspect of the formed tissues in all scaffolds resembled more connective tissue than a dentin-like tissue. Limited calcification of the ECM was only seen in the ceramic scaffold. In both experiments, no other differences could be attributed to the different materials used. In conclusion, the in vivo behavior of DPSC and their relations with 3-D scaffold materials should be further studied before clinical use can be considered.     

Soluble factor-dependent in vitro growth and maturation of rat fetal liver cells in a three-dimensional culture system

Fetal liver cell fractions are potent sources of cells for future liver tissue engineering, by virtue of their high proliferation capacity and their potential for hepatic maturation. Recently, some types of hepatic differentiation agents have been identified from findings in stem cell biology. We therefore investigated the in vitro growth and maturation of rat fetal liver cells isolated from 17-day-old pregnant rats in poly-L-lactic acid three-dimensional (3D) macroporous scaffolds in the presence of soluble factors, such as a combination of hepatocyte growth factor, fibroblast growth factor-1, and fibroblast growth factor-4, oncostatin M, and sodium butyrate. Inclusion of all these factors in the 3D culture induced higher levels of hepatic functions and well maintained these enhanced levels during 2 weeks of culture, whereas in the monolayer culture, such functional enhancement was gradually lost after 1 week. The finally achieved functions on a per-cell basis in the 3D culture with all of the soluble factors were comparable to those of adult hepatocytes. We therefore conclude that the 3D culture system shows promise for the in vitro maturation of fetal liver cells as a means of preconditioning of the cells for engineered liver tissue equivalents in future transplantation studies.     

Development of an esophagus acellular matrix tissue scaffold

A cell-extraction protocol yielding an esophagus acellular matrix (EAM) scaffold for use in tissue engineering of an esophagus, including hypotonic lysis, multiple detergent cell extraction steps, and nucleic acid digestion, was developed in a rat model. Histological techniques, burst pressure studies, in vitro esophageal epithelial cell seeding, and in vivo implantation were used to assess cell extraction, extracellular matrix (ECM) preservation, and biocompatibility. Microscopy demonstrated that cell extraction protocols using sodium dodecyl sulfate (SDS) (0.5%, wt/vol) as a detergent resulted in cell-free EAM with retained ECM protein collagen, elastin, laminin, and fibronectin. Burst pressure studies indicated a loss of tensile strength in EAMs, but at intraluminal pressures that were unlikely to affect in vivo application. In vitro cell seeding studies exhibited epithelial cell proliferation with stratification similar to native esophagi after 11 days, and subcutaneously implanted EAMs displayed neovascularization and a minimal inflammatory response after 30 days of implantation. This study presents an esophagus acellular matrix tissue scaffold with preserved ECM proteins, biomechanical properties, and the ability to support esophageal cell proliferation to serve as the foundation for a tissue-engineered esophagus.     

Application of cell technologies to tissue defect closure in oncology

In vivo and in vitro experiments on the application of cell technologies to tissue defect closure were conducted; autologic mesenchymal stem cells on 3-demensional matrices were used. The authors analyze the results of the application of bioengineering tissue equivalents for the closure of soft tissue and upper airway defects after extensive resections performed in 52 oncological patients. Tissue equivalents with stem cells provide engraftment and long-term graft functioning; they also modify wound surface, thus stimulating wound epithelization. In this study the application of tissue equivalents led to wound healing and functional recovery in 87% of patients.     

Three-dimensional hydrogel scaffolds facilitate in vitro self-renewal of human skin-derived precursors

Skin-derived precursors (SKPs) are multipotent cells with dermal stem cell properties. These easily available cells possess the capacity to reconstitute the skin in vivo, as well as a broader differentiation potential in vitro, which endows them with great prospects in regenerative medicine. However, the present authors’ group and others previously found that adult human SKPs (hSKPs) expanded deficiently in vitro, which largely counteracted their research and practical values. Taking the physiological micro-environment of hSKPs into consideration, the authors sought to establish a hydrogel scaffold-based three-dimensional (3-D) culture system for hSKPs in the present study. After comparing their morphology, growth characteristics, signature gene expression and differentiation potential in different hydrogels, the present authors found that a chemically defined hyaluronic acid and denatured collagen-based hydrogel system that mimicked the natural niche of hSKPs in the dermis could alleviate hSKP senescence, support hSKP proliferation as spheres, while largely retaining their properties and potential. This study suggested that recapitulating the in vivo stem cell niche by providing them with 3-D extracellular matrix environments could help them achieve better self-renewal in vitro. In addition, the animal-origin-free and biocompatible 3-D hydrogel system will certainly benefit fundamental research and clinical applications of hSKPs in the near future.     

A 3-D organoid kidney culture model engineered for high-throughput nephrotoxicity assays

Cell-cell and cell-matrix interactions control cell phenotypes and functions in vivo. Maintaining these interactions in vitro is essential to both produce and retain cultured cell fidelity to normal phenotype and function in the context of drug efficacy and toxicity screening. Two-dimensional (2-D) cultures on culture plastics rarely recapitulate any of these desired conditions. Three dimensional (3-D) culture systems provide a critical junction between traditional, yet often irrelevant, in vitro cell cultures and more accurate, yet costly, in vivo models. This study describes development of an organoid-derived 3-D culture of kidney proximal tubules (PTs) that maintains native cellular interactions in tissue context, regulating phenotypic stability of primary cells in vitro for up to 6 weeks. Furthermore, unlike immortalized cells on plastic, these 3-D organoid kidney cultures provide a more physiologically-relevant response to nephrotoxic agent exposure, with production of toxicity biomarkers found in vivo. This biomimetic primary kidney model has broad applicability to high-throughput drug and biomarker nephrotoxicity screening, as well as more mechanistic drug toxicology, pharmacology, and metabolism studies.     

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.     

Proteins combination on PHBV microsphere scaffold to regulate Hep3B cells activity and functionality: a model of liver tissue engineering system

The synergistic effects of extracellular matrix (ECM) protein combinations on Hep3B cell proliferation and functions are studied herein. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres were covalently conjugated with three types of proteins, collagen (type I), laminin, and fibronectin, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide cross linkers. Successful conjugations of protein molecules were verified by the presence of nitrogen peaks in X-ray photoelectron spectroscopy. The densities of grafted proteins were quantified using Micro-BCA kit. A human hepatoma cell line, Hep3B, was then cultured in vitro on the ECM proteins-modified microspheres for 2 weeks. Cell proliferation was estimated using MTT method, and two hepatic functions, albumin secretion and P-450 activity, were evaluated using ELISA and EROD assays, respectively. The results indicated that combination of the three ECM proteins on microsphere surfaces has a significant effect on the proliferation of Hep3B cells, thus better mimicking the in vivo environment for liver tissue engineering.     

Tissue vascularization through 3D printing: Will technology bring us flow?

Background: Though in vivo models provide the most physiologically relevant environment for studying tissue function, in vitro studies provide researchers with explicit control over experimental conditions and the potential to develop high throughput testing methods. In recent years, advancements in developmental biology research and imaging techniques have significantly improved our understanding of the processes involved in vascular development. However, the task of recreating the complex, multi‐scale vasculature seen in in vivo systems remains elusive. Results: 3D bioprinting offers a potential method to generate controlled vascular networks with hierarchical structure approaching that of in vivo networks. Bioprinting is an interdisciplinary field that relies on advances in 3D printing technology along with advances in imaging and computational modeling, which allow researchers to monitor cellular function and to better understand cellular environment within the printed tissue. Conclusions: As bioprinting technologies improve with regards to resolution, printing speed, available materials, and automation, 3D printing could be used to generate highly controlled vascularized tissues in a high throughput manner for use in regenerative medicine and the development of in vitro tissue models for research in developmental biology and vascular diseases. Developmental Dynamics 244:629–640, 2015. © 2015 Wiley Periodicals, Inc.     

Morphology and function of ovine articular cartilage chondrocytes in 3-d hydrogel culture

Different cell- and biomaterial-based tissue engineering techniques are under investigation to restore damaged tissue. Strategies that use chondrogenic cells or tissues in combination with bioresorbable delivery materials are considered to be suitable to regenerate bio-artificial cartilage. Three-dimensional (3-D) cell embedding techniques can provide anchorage-independent cell growth and homogenous spatial cell arrangement, which play a key role in the maintenance of the characteristic phenotype and thus the formation of differentiated tissue. We developed a new injectable high water content (90%) hydrogel formulation with 5% sodium alginic acid and 5% gelatin as a temporary supportive intercellular matrix for 3-D cell culture. The objective was to determine whether the in vitro hydrogel culture of chondrocytes could preserve hyaline characteristics and thus could provide cartilage regeneration in vitro. Chondrocytes harvested from knee joints of skeletally mature sheep were cultured 3-D in hydrogel (7 x 10(6) cells/ml, 2.8-mul beads) for up to 10 weeks. Cell morphology and viability were evaluated with light microscopy, and proliferative activity was assessed with antibromodeoxyuridine immunofluorescence. Expression of collagens type I (COL1) and II (COL2), cartilage proteoglycans (PG) and hyaluronan synthases (HAS) were studied immunohistochemically. We observed that up to 36% of chondrocytes proliferated, while almost 100% presented a differentiated spheroidal phenotype. After an initial decrease at 2 weeks, cell density recovered to 85% of the initial absolute value at 10 weeks. Expression of hyaline matrix molecules resembled the in vivo pattern with increasing spatial deposition of PG and COL2. The proportion of PG-positive cells increased from initially 13 to 53% after 10 weeks, in contrast to consistently 100% COL2-positive cells. We conclude that 3-D hydrogel culture, even without mechanical stimulation or growth factor application, can keep chondrocytes in a differentiated state and provides a chondrogenic cell environment for in vitro cartilage regeneration for at least 10 weeks. Moreover, this hydrogel appears to be a suitable cell delivery material for subsequent in vivo implantation.     

Optimization of an in vitro three-dimensional microenvironment to reprogram synovium-derived stem cells for cartilage tissue engineering

Adult stem cells gradually lose their stemness when plated in monolayer culture after isolation from their in vivo niche. In this study, we hypothesized that the in vitro microenvironment can be optimized by modulating oxygen tension and mitotic signal in a tissue-specific extracellular matrix (ECM) deposited by synovium-derived stem cells (SDSCs) to rejuvenate expanded SDSC proliferation and chondrogenic potential. Passage 3 SDSCs were plated on either SDSC-derived ECM or plastic flask and incubated in either hypoxia (5% O(2)) or normoxia (21% O(2)) with or without the supplementation of 10?ng/mL of basic fibroblast growth factor-2 (FGF-2) for 7 days, followed by pellet culture in a serum-free chondrogenic medium for 14 days. Our data showed that, compared with the mitotic effect of FGF-2 on SDSCs, ECM expansion greatly enhanced SDSC proliferation while retaining SDSC stem cell characteristics. More importantly, ECM pretreatment yielded SDSC pellets with a comparable chondrogenic index to FGF-2 pretreatment, both of which were much higher than SDSC expansion on plastic flask alone. FGF-2 pretreatment led to the highest glycosaminoglycans and DNA content; intriguingly, it also contributed to the highest expression level of hypertrophic marker genes. Surprisingly, the hypertrophic marker genes could be downregulated if the pretreatment was combined with hypoxia or ECM. The combination of hypoxia, FGF-2, and SDSC-derived ECM contributed to the highest cell number in SDSC expansion. Our study indicates that the three-dimensional microenvironment for ex vivo expansion can be optimized to provide high-quality stem cells for stem cell-based cartilage defect repair.     

Inhibition effect of Oncostatin M on metastatic human lung cancer cells 95-D in vitro and on murine melanoma cells B16BL6 in vivo

Oncostatin M (OSM) is a multifunctional regulator of cell growth and differentiation. It inhibits the growth of many types of tumor cells, but its role in metastasis is unknown. We studied the human OSM expressed and purified from reconstructed E. Coli on its activity of inhibiting metastasis of tumor cells by a series of assays in vitro and in vivo. Clone formation assay in soft agar was used to measure the inhibition activity of OSM on the proliferation of high metastatic human lung cancer cells 95-D. Cell attachment assay, cell migration assay and cell invasion assay were used to evaluate inhibition by OSM on 95-D cells of the adhesion ability, the migration ability, and the ability of cells to cross tissue barriers, respectively. Inhibition of OSM on secretion of MMP-2 and -9 secretion in 95-D cells was determined by Western blot. The in vivo inhibitory effect of OSM on metastasis of murine melanoma cells B16BL6 was examined in the pulmonary metastasis model. In vitro studies showed that OSM inhibited the proliferation of 95-D cells at low concentration. OSM also reduced the adhesion and invasion ability of 95-D cells and inhibited the secretion of MMP-2 and MMP-9 in OSM treated cells. In vivo results showed that OSM (20 microg/kg/d for 7 days) inhibited pulmonary metastasis at a rate of 20.7%. There were no differences in animal weights among the groups. These results suggest that OSM has the potential of being a clinical inhibitor on metastasis of some cancer cells.