The effect of cell passage number on osteogenic and adipogenic characteristics of D1 cells.

Cell line passage number is an important consideration when designing an experiment. At higher passages, it is generally understood that cell health begins to decline and, when this occurs, the result can be variable data. However, there are no specific guidelines regarding optimal passage range, and this information is dependent on cell type. To explore these variabilities, low passage D1 cells were thawed (passage 3) and passaged serially until a much higher number (passage 34). Samples were taken every five passages and analyzed for alkaline phosphatase and triglyceride; also, the gene expression of both adipogenic and osteogenic markers was tested. The results indicate that the growth rate of these cells did slow down after passage 30. However, expression of the osteogenic characteristics seemed to cycle, with the highest levels seen at passage 4 and 24. The adipocyte expression levels remained the same throughout the study.

Designing a tunable 3D heterocellular breast cancer tissue test system.

It is increasingly recognized that the tissue microenvironment is crucial in cell signaling and regulation of normal and malignant cell function. Components and properties of the microenvironment such as extracellular matrix, adhesion integrins, tissue architectures and tissue modulus regulate growth, differentiation and apoptosis of cells. These properties control cell fate through complex signals that are influenced either by interactions between neighbouring cells or by stimulated cell surface receptors. In this study, we established an in vitro engineered microenvironment: i.e., a tissue test system that combined heterocellular tumour spheroids, polymeric microcarriers and adipocytes, an abundant stromal cell type in breast tissue, to investigate the behaviour of breast cancer cells in response to different environmental stimuli in a more relevant 3D microenvironment. Results showed the engineered microenvironment influenced breast cancer cell proliferation, differentiation and migration through multi-cellular interactions and changes in microenvironmental stiffness and that stromal cells such as adipocytes play a critical role in the breast cancer process.

Effect of ceramide on mesenchymal stem cell differentiation toward adipocytes.

Proinflammatory cytokines such as tumor necrosis factor (TNF) alpha are well known to inhibit adipocyte differentiation. TNF-alpha triggers ceramide synthesis through binding of TNF-alpha to its p55 receptor. Therefore, ceramide is implicated in many of the multiple signaling pathways initiated by TNF-alpha. In breast tissue engineering, it is important to know how to modulate adipocyte differentiation of the stem cells with exogenous additives like ceramide in vitro. We hypothesized that stem cell adipogenesis could be retained in TNF-alpha-treated preadipocytes in which ceramide synthesis was blocked and that exogenous ceramide could inhibit adipocyte differentiation. We first studied the effect of ceramide synthase inhibitor, Fumonisin B2, on the adipogenesis of murine mesenchymal stem cells (D1 cells), treated with TNF-alpha. We then studied the effect of specific exogenous C6-ceramide on D1 cell viability and differentiation. It was found that 1 ng/ml of TNF-alpha significantly inhibited D1 cell adipogenesis. Cells treated with 5 microM of Fumonisin B2 were able to undergo adipogenesis, even when treated with TNF-alpha. High concentrations of exogenous C6-ceramide (>50 microM) had an inhibitory effect, not only on the pre-confluent proliferation of the D1 cells but also on the post-confluent cell viability. High concentrations of C6-ceramide (>50 microM) also inhibited mitotic clonal expansion when D1 cell differentiation was induced by the addition of an adipogenic hormonal cocktail. C6-ceramide at low concentrations (10-25 microM) inhibited lipid production in D1 cells, demonstrated by decreased levels of both total triglyceride content and specific fatty acid composition percentages. Genetic expression of peroxisome proliferator-activated receptor (PPAR) gamma and aP2 in D1 cells was reduced by C6-ceramide treatment. CCAAT/enhancer-binding protein (C/EBP) beta levels in D1 cells were reduced by C6-ceramide treatment during early differentiation; PPARgamma and aP2 protein levels were reduced at terminal differentiation. C6-ceramide at lower concentrations also decreased lipid accumulation of differentiating D1 cells. Our results suggest that ceramide synthase inhibitor retains the adipogenic potential of TNF-alpha-treated mesenchymal stem cells, while exogenous ceramide at lower concentrations inhibit the adipogenesis of mesenchymal stem cells. Ceramide, therefore, could be a modulator candidate in breast tissue engineering strategies.

Application of magnetic resonance microscopy to tissue engineering: a polylactide model.

Absorbable polymers are unique materials that find application as temporary scaffolds in tissue engineering. They are often extremely sensitive to histological processing and, for this reason, studying fragile, tissue-engineered constructs before implantation can be quite difficult. This research investigates the use of noninvasive imaging using magnetic resonance microscopy (MRM) as a tool to enhance the assessment of these cellular constructs. A series of cellular, polylactide constructs was developed and analyzed using a battery of tests, including MRM. Distribution of rat aortic smooth muscle cells within the scaffolds was compared as one example of a tissue engineering MRM application. Cells were loaded in varying amounts using static and dynamic methods. It was found that the cellular component was readily identified and the polymer microstructure readily assessed. Specifically, the MRM results showed a heterogeneous distribution of cells due to static loading and a homogenous distribution associated with dynamic loading, results that were not visible through biochemical tests, scanning electron microscopy, or histological evaluation independently. MRM also allowed differentiation between different levels of cellular loading. The current state of MRM is such that it is extremely useful in the refinement of polymer processing and cell seeding methods. This method has the potential, with technological advances, to be of future use in the characterization of cell-polymer interactions.

Absorbable mesh aids in development of discrete, tissue-engineered constructs.

Absorbable mesh was investigated as a potential containment material in which to house discrete, small, tissue-engineered constructs. The mesh was fashioned into bags of varying shapes and consistent volumes. Cells were cultivated on porous, collagen beads, and the tissue constructs were placed into the bags. The mechanical integrity of the bags and feasibility of the design was tested in vitro. The bags successfully maintained their integrity as the cells developed on the collagen matrices. Furthermore, their porosity allowed access of nutrients and waste products to and from the developing tissue. Having demonstrated feasibility of processing, the next step is to optimize the cell culture specifications and materials design.

Biomaterial developments for bone tissue engineering.

The development of bone tissue engineering is directly related to changes in materials technology. While the inclusion of materials requirements is standard in the design process of engineered bone substitutes, it is also critical to incorporate clinical requirements in order to engineer a clinically relevant device. This review presents the clinical need for bone tissue-engineered alternatives to the present materials used in bone grafting techniques, a status report on clinically available bone tissue-engineering devices, and recent advances in biomaterials research. The discussion of ongoing research includes the current state of osseoactive factors and the delivery of these factors using bioceramics and absorbable biopolymers. Suggestions are also presented as to the desirable design features that would make an engineered device clinically effective.

Comparative study of seeding methods for three-dimensional polymeric scaffolds.

Development of tissue-engineered devices may be enhanced by combining cells with porous absorbable polymeric scaffolds before implantation. The cells are seeded throughout the scaffolds and allowed to proliferate in vitro for a predetermined amount of time. The distribution of cells throughout the porous material is one critical component determining success or failure of the tissue-engineered device. This can influence both the successful integration of the device with the host tissue as well as the development of a vascularized network throughout the entire scaffold volume. This research sought to compare different seeding and proliferation methods to select an ideal method for a polyglycolide/aortic endothelial cell system. Two seeding environments, static and dynamic, and three proliferation environments, static, dynamic, and bioreactor, were analyzed, for a total of six possible methods. The six seeding and proliferation combinations were analyzed following a 1-week total culture time. It was determined that for this specific system, dynamic seeding followed by a dynamic proliferation phase is the least promising method and dynamic seeding followed by a bioreactor proliferation phase is the most promising.

The development of an embedding technique for polylactide sponges.

The use of absorbable polymeric biomaterials is increasing in the field of tissue engineering. These polymeric scaffolds provide mechanical strength and shape as the engineered tissue forms. Histological analysis is an important part of the development of an appropriate polymeric construct, because it allows the analysis of the cell/material interaction. Unfortunately, routine paraffin processing often degrades these absorbable polymers, and routine staining can dissolve the remnants. This research sought to develop a histological procedure that would retain the polymer structure. Two processing procedures, paraffin and glycol methacrylate, were tested on three in vitro groups of poly-L-lactide sponges, high cell density seeding, low cell density seeding, and a control. The paraffin processing caused shrinkage and degradation of the polymer, and staining dissolved the remnants. The glycol methacrylate processing minimized damage to the polymer even after staining.

Parameters affecting cellular adhesion to polylactide films.

Absorbable biomaterials have been recently incorporated into the field of tissue engineering. Little work has been performed, even with the clinically acceptable absorbables, concerning their tissue promoting capability or lack, thereof. Furthermore, the relative attractions of cells to these implants may be largely disguised by the presence of serum. This research involved the development of an adhesion assay to compare the adhesion behavior of two cell types to two different polylactides in a serum free environment. The results showed that the attachment behavior depends not only on the cell or the polymer but a combination of the two.

Cellular ingrowth and thickness changes in poly-L-lactide and polyglycolide matrices implanted subcutaneously in the rat.

Highly porous matrices of poly-L-lactide (PL) and polyglycolide (PG), 24, 50, or 95 mg/cc in the form of 10 x 10 x 3 mm wafers, were implanted subcutaneously (two per rat) in the flanks of 8-12-week-old female Lewis rats (n = 120). Matrices were harvested, two rats per week, for 15 weeks and examined histologically. At weeks 1 and 2, a thin fibrous capsule was present and matrices showed capillary beds and host-cell infiltration along the implant margins. By week 4, the PL specimens had some arterioles while the PG specimens still had only capillary beds. At week 7, PL had well developed arterioles, venules, and capillaries while PG began to show modest vascular beds of capillaries only. In terms of cellular ingrowth, PL remained unchanged from 7 to 15 weeks. Giant cell formation was observed wherever polymer was present. There was a loss of thickness and cell mass for both matrices over time (PG > PL) despite initial host-cell ingrowth. As both polymers degraded and were absorbed, the ingrown cells mass regressed. There was little remaining PG at 15 weeks, leaving no trace of cells that previously had ingrown and no evidence of scar tissue.

Change in stiffness and effect of orientation in degrading polylactide films.

The degradation pattern of the synthetic absorbable polyester is thought to occur from the center of the material outward, and the bulk degradation is therefore attributed largely to the chemical composition of the material. It was hypothesized that this pattern might be altered by changing the morphology of the material, i.e., by introducing molecular orientation into the system. A new solid state uniaxial orientation (SS-UO) process was used to orient two types of lactide polymer films. The films were exposed to a phosphate buffered solution, then chemically, mechanically, and visually analyzed after predetermined times. This paper explores the results of flexural testing which will be later correlated with microscopic degradation events, as part of the larger degradation study. The results show that, while orientation does not have an overall significant effect on the flexural modulus, there is a significant material/orientation interaction.

Development of technologies aiding large-tissue engineering.

There are many clinical situations in which a large tissue mass is required to replace tissue lost to surgical resection (e.g., mastectomy). It is possible that autologous cell transplantation on biodegradable polymer matrices may provide a new therapy to engineer large tissue which can be used to treat these patients. A number of challenges must be met to engineer a large soft tissue mass. These include the design of (1) a structural framework to maintain a space for tissue development, (2) a space-filling matrix which provides for localization of transplanted cells, and (3) a strategy to enhance vascularization of the forming tissue. In this paper we provide an overview of several technologies which are under development to address these issues. Specifically, support matrices to maintain a space for tissue development have been fabricated from polymers of lactide and glycolide. The ability of these structures to resist compressive forces was regulated by the ratio of lactide to glycolide in the polymer. Smooth muscle cell seeding onto polyglycolide fiber-based matrices has been optimized to allow formation of new tissues in vitro and in vivo. Finally, polymer microsphere drug delivery technology is being developed to release vascular endothelial growth factor (VEGF), a potent angiogenic molecule, at the site of tissue formation. This strategy, which combines several different technologies, may ultimately allow for the engineering of large soft tissues.

Physicochemical changes in degrading polylactide films.

It has been suggested in the literature that 'large' size bioabsorbable aliphatic polyester devices degrade heterogeneously when exposed to an aqueous environment. That is, following saturation, the material degrades preferentially from the center to the exterior due to an auto catalytic effect. Oriented absorbable films were developed using a new solid state method in order to assess the influence of molecular orientation on degradation pattern. The method entails uniaxial deformation and thus is referred to as solid state uniaxial orientation (SS-UO). This work examines solely the physicochemical changes occurring in the degrading polylactide film and their relevance to changes in key molecular parameters, as part of a broad based study on the effect of orientation on absorption. The results indicate that the orientation has a large effect on the glass transition temperature and the heat of fusion.

Water fugacity in absorbing polymers.

Absorbable biomaterials, as dynamic systems, require special handling, processing, and characterization techniques beyond those of the traditional nonabsorbable materials. As the material degrades or absorbs, in vitro or in vivo, it undergoes structural, physical, and chemical changes. These changes in the base material may significantly impact the performance of a particular biomedical device; hence, it is important that the investigator consider the full range of properties that constitute the lifetime of a given absorbable material. The long term degradation study presented here sought to identify one such property, the change in water retention of a degrading oriented polylactide film. The investigation found through differential scanning calorimetry that later stages of degradation are often characterized by a stronger retention of water, potentially due to a higher number of polar carboxyl groups within the relatively hydrophobic polymer matrix.

Modulation of surface and bulk properties of biomedical polymers.

The surface and bulk modulation of polymeric biomedical devices allows the full range of material properties to be exercised as demanded by custom applications. Polymeric biomaterials are finding greater use as relatively inert and even transient options and so therefore will require thorough processing analyses and the transfer of technology from nonbiomedical applications to the biomedical industry.