Natural polymers are widely used in a variety of biomedical applications including, tissue regeneration, drug delivery, and for imaging. In wound care and tissue regeneration, they are used as dressings for acute or chronic wounds and as regeneration templates. Prosthetic mesh material can be used to induce the repair and restoration of soft tissues following surgery and deliver stem cells to damaged tissue.  Common features of this extracellular matrix-assisted tissue remodeling include promotion of angiogenesis, recruitment of circulating progenitor cells and constructive remodeling of damaged tissue structures. Natural polymers can be derived from a wide variety of sources, from plants, animals, and microorganisms. Natural polymers that mimic human extracellular matrix have mechanical tunability, high biocompatibility, and high-water holding capacity. The extracellular matrix is comprised of many polymers, including proteoglycans, hyaluronic acid, collagen and elastin. Natural polymers–based scaffolds are appealing for localized drug and stem cell delivery, tissue repair and regeneration. Careful characterization and selection of the appropriate biopolymer material is particularly important. Here we discuss properties and characterization of natural polymers with a specific focus on Collagen.

Collagen, the most abundant extracellular matrix protein in animal kingdom belongs to a family of fibrous proteins, which give strength and flexibility to tissues and provide a highly biocompatible environment for cells. Collagen is an ideal biomaterial for implantable medical products and scaffolds for in vitro testing systems. To manufacture collagen-based solutions, porous sponges, membranes and threads for surgical and dental purposes or cell culture matrices, collagen rich tissues as skin and tendon of mammals are intensively processed by physical and chemical means. Other tissues such as pericardium and intestine are more gently decellularized while maintaining their complex collagenous architectures. New processing technologies have generated structurally versatile materials controlling for strength, stability, and biocompatibility. Important progress is expected in this field and will aide in the advancement of biopharmaceuticals, tissue regeneration and cell therapy. The extracellular matrix is comprised of many biopolymers and is complex and difficult to study. Characterization of biopolymers, including collagen, is important for successful therapeutic and device development.

Quality control testing involves measures of safety and characterization of the natural polymer. These tests include sterility, endotoxin, DNA content, and residual chemicals from processing. Physical and chemical characterization may include measurement of tensile strength, polymer cross linking, and total protein content.  Identification of the natural polymer derived products includes testing for proteoglycans, growth factors and cytokines. Demonstrating quality and efficacy of natural polymers by functional testing such as cell migration, stem cell survival or apoptosis, or lineage differentiation.

Cell Migration is measured using Transwell chambers with different conditions to assess inhibitors and attractant’s ability to alter cell movement.  Polymer products can be tested for their ability to promote cell survival using live/dead stains or measures of apoptotic signaling. Degradation of collagen fibers is assessed by measuring N-terminal propeptide or hydroxyproline residues by ELISA in complex matrices. Thermal stability studies measure degradation using differential scanning calorimetry (DSC). Collagen binding dyes or peptides that bind selectively to denatured collagen are also useful. For example, a trichrome histology stain is a mixture of three dyes used on connective tissue to visualize collagen and reticular fibers. Collagen fibers stain green or blue with Masson's trichrome stain. Muscle and keratin stain red. Sirius Red stains native total collagen (all types). Much can be learned by observing the morphology of the polymer product with microscopic examination. Modern imaging techniques such as MRI and PET are valuable tools to image connective tissue.

Natural polymers, especially collagens and other components of the extracellular matrix play critical roles in health and disease. Defects in extracellular matrix may cause bone and connective tissue disorders, cardiovascular disease, and dermatological conditions. More common types of connective tissue disorders include arthritis, fibromyalgia, scleroderma, and lupus. Fibrotic conditions (abnormal deposition of extracellular matrix) underlie many major diseases. The importance and utility of natural polymers for biomedical applications is clear. The use and characterization of natural polymers is an active field of study that requires special expertise and analytical support.

Biomaterials and polymers are increasingly combined with drugs and biologics. Regenerative products may have medical device–based scaffolding and may be treated as biologics, reflecting the cell and tissue components. Therefore, biomaterials, medical device components, and 3-D printed materials need to be fully characterized for biocompatibility. Biological reactivity tests (see USP <87> and ISO 10993-5 guidances) are designed to determine the biocompatibility of mammalian cell cultures following contact with elastomeric plastics and other polymeric materials with direct or indirect patient contact or of specific extracts prepared from the materials under test.

For medical devices, all biocompatibility and efficacy testing occurs prior to any clinical testing. Being aware of and applying the proper testing standards to development and testing of the device can facilitate entry into the desired national or global market place.  Medical devices are characterized using cell culture assay to assess the biocompatibility of a material or extract through the use of mammalian cells. These techniques provide an excellent way to screen materials prior to expensive animal testing.

Per the USP <87> monograph, there are three cytotoxicity tests commonly used for medical devices. The direct contact test is designed for materials in a variety of shapes. The procedure allows for simultaneous extraction and testing of leachable chemicals from the specimen with a serum-supplemented medium. The procedure is not appropriate for very low- or high-density materials that could cause mechanical damage to the cells. Reactivity of the test sample is indicated by changes in morphology, membrane degeneration and lysis of cells around the test material.

The agar diffusion test is designed for elastomeric closures in a variety of shapes. The agar layer acts as a cushion to protect the cells from mechanical damage while allowing the diffusion of leachable chemicals from the polymeric specimens.

The MEM Elution test is designed for the evaluation of extracts of polymeric materials. The procedure allows for extraction of the specimens at physiological or non-physiological temperature. This test may use different extraction media and extraction conditions to test devices according to actual use conditions or to exaggerate those conditions.  After preparation, the extracts are transferred onto a layer of cells (typically mouse fibroblast L-929 cells).  Following incubation, the cells are examined microscopically for morphology and lysis of the cells.

Recent regulations (ANSI/AAMI/ISO 10993-5:2009) on biocompatibility for devices state that the three qualitative cytotoxicity tests are appropriate for screening purposes, but that quantitative evaluation is recommended.  There are several quantitative cell viability assays available such as colorimetric MTT assay, FL staining for live/dead cells, or luciferase based cell viability testing. Our laboratory has cell imaging capabilities that include multi-color Fluorescence (using the Thermo Evos cell imaging platform) or plate based quantitative spectrophotometric analysis using UV/Vis, FL, and luminescence detection (using Molecular Devices platforms). Cell cytotoxicity assays are just one way we can characterize your medical device, or biopharmaceutical product.