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.

A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep.

Soft tissue reconstruction using tissue-engineered constructs requires the development of materials that are biocompatible and support cell adhesion and growth. The objective of this study was to evaluate the use of macroporous hydrogel fragments that were formed using either unmodified alginate or alginate covalently linked with the fibronectin cell adhesion peptide RGD (alginate-RGD). These materials were injected into the subcutaneous space of adult, domesticated female sheep and harvested for histological comparisons at 1 and 3 months. In addition, the alginate-RGD porous fragments were seeded with autologous sheep preadipocytes isolated from the omentum, and these cell-based constructs were also implanted. The results from this study indicate that both the alginate and alginate-RGD subcutaneous implants supported tissue and vascular ingrowth. Furthermore, at all time points of the experiment, a minimal inflammatory response and capsule formation surrounding the implant were observed. The implanted materials also maintained their sizes over the 3-month study period. In addition, the alginate-RGD fragments supported the adhesion and proliferation of sheep preadipocytes, and adipose tissue was present within the transplant site of these cellular constructs, which was not present within the biomaterial control sites.

In vivo characterization of a porous hydrogel material for use as a tissue bulking agent.

Tissue engineered biomaterial constructs are needed for plastic and reconstructive applications. To successfully form a space-filling tissue, the construct should induce a minimal inflammatory response, create minimal or no fibrotic capsule, and establish a vascular bed within the first few days after implantation to ensure survival of the implanted cells. In addition, the biomaterial should support cellular adhesion and induce tissue ingrowth. A macroporous hydrogel bead using sodium alginate covalently coupled with an arginine, glycine, and aspartic acid-containing peptide was created. A 6-month subcutaneous rat model study was performed to determine if the implanted material induced tissue ingrowth throughout the implantation area and maintained a three-dimensional vascular bed. The implanted materials produced a vascular bed, minimal inflammation and capsule formation, and good tissue ingrowth throughout the experiment. The material retained its bulking capacity by demonstration of no significant change of the cross-sectional area as measured from the center of the implants after the 2-week time point. In addition, the granulation tissue formed around the implant was loosely organized, and the surrounding tissue had integrated well with the implant. These results indicate that this material has the desired properties for the development of soft-tissue-engineering constructs.

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.

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.

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.