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1.1 This guide covers an overview of test methods that may be used to obtain information relating to the dimensions of pores, the pore size distribution, the degree of porosity, interconnectivity, and measures of permeability for porous materials used as polymeric scaffolds in the development and manufacture of tissue-engineered medical products (TEMPs). This information is key to optimizing the structure for a particular application, developing robust manufacturing routes, and providing reliable quality control data.

1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.

1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.

1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

5.1 The ability to culture functional tissue to repair damaged or diseased tissues within the body offers a viable alternative to xenografts or heterografts. Using the patient’s own cells to produce the new tissue offers significant benefits by limiting rejection by the immune system. Typically, cells harvested from the intended recipient are cultured in vitro using a temporary housing or scaffold. The microstructure of the scaffold can be defined by the existence, type, size distribution, interconnectivity, and directionality of pores – all of which are critical for cell migration, growth, and proliferation (Appendix X1). Optimizing the design of tissue scaffolds is a complex task, given the range of available materials, different manufacturing routes, and processing conditions. All of these factors can, and will, affect the surface texture, surface chemistry, and microstructure of the resultant scaffolds. Surface texture, surface chemistry, and microstructure of the scaffolds may or may not be significant variables depending on the characteristics of a given cell type at any given time (that is, changes in cell behavior due to the number of passages, mechanical stimulation, and culture conditions).

5.2 Tissue scaffolds are typically assessed using an overall value for scaffold porosity and a range of pore sizes, though the distribution of sizes is rarely quantified. Published mean pore sizes and distributions are usually obtained from electron microscopy images and quoted in the micrometer range. Tissue scaffolds are generally complex structures that are not easily interpreted in terms of pore shape and size, especially in three dimensions. Therefore, it is difficult to quantifiably assess the batch-to-batch variance in microstructure or to make a systematic investigation of the role that the mean pore size and pore size distribution has on influencing cell behavior based solely on electron micrographs (Tomlins et al, (1)).4

5.2.1 Fig. 1 gives an indication of potential techniques that can be used to characterize the structure of porous tissue scaffolds and the length scale that they can measure. Clearly a range of techniques must be used if the scaffold is to be characterized in detail.

5.2.2.1 Since the literature contains many other terms for defining pores (Perret et al (3)), it is recommended that the terms used by authors to describe pores be defined in order to avoid potential confusion. Additionally, since any of the definitions in Table 2 can shift, depending on the pore size determination method (see Table 1 and Fig. 1), an accompanying statement describing the assessment technique used is essential.

FIG. 2 A through-pore showing a variation of pore diameter, D (a); and an example of a blind-pore (b).

5.2.3 All the techniques listed in Table 1 have limitations for assessing complex porous structures. Fig. 2a and Fig. 2b show a through- and a blind-end pore respectively. Porometry measurements (see 7.4) are only sensitive to the narrowest point along a variable diameter through-pore and therefore can give a lower measure of the pore diameter than other investigative techniques, such as scanning electron microscope (SEM), which may sample at a different point along the pore. The physical basis of porometry depends on the passage of gas through the material. Therefore, the technique is not sensitive to blind-end or closed pores. Estimates of porosity based on porometry data will therefore be different from those obtained from, for example, porosimetry (see 7.3), which is sensitive to both through- and blind-pores or density determinations that can also account for through-, blind-end, and closed pores. The significance of these differences will depend on factors such as the percentage of the different pore types and their dimensions. Further research will enable improved guidance to be developed.

5.2.4 Polymer scaffolds range from mechanically rigid structures to soft hydrogels. The methods currently used to manufacture these structures include, but are not limited to:

5.2.4.1 Casting a polymer, dissolved in an organic solvent, over a water-soluble particulate porogen, followed by leaching.

5.2.4.2 Melt mixing of immiscible polymers followed by leaching of the water-soluble component.

5.2.4.3 Dissolution of supercritical carbon dioxide under pressure into an effectively molten polymer, a phenomenon attributed to the dramatic reduction in the glass transition temperature which occurs, followed by a reduction in pressure that leads to the formation of gas bubbles and solidification.

5.2.4.4 Controlled deposition of molten polymer to produce a well-defined three-dimensional lattice.

5.2.4.5 The manufacture of three-dimensional fibrous weaves, knits, or non-woven structures.

5.2.4.6 Chemical or ionic cross-linking of a polymeric matrix.

5.2.5 Considerations have been given to the limitations of these methods in Appendix X1.

5.2.6 This guide focuses on the specific area of characterization of polymer-based porous scaffolds and is an extension of an earlier ASTM guide, Guide F2150.