Bacterial Cellulose Scaffolds and Cellulose Nanowhiskers

Biocompatibility of Cellulose

While specific examples of biomedical applications of nanocellulose fibers and, in particular, tissue engineering, will be given in the following sections, a brief discussion of some earlier general studies into the biocompatibility of cellulose both in vitro and in vivo is called for. Indeed, unlike some common biomaterials, such as collagen hydrogels and scaffolds, the nonanimal origin of cellulosic materials leads to reasonable questions about both biocompatibility and biodegradation. A small number of studies have been published over the last 20 years, with the aim of addressing these questions using a variety of materials and techniques.

In an early in vivo study, Miyamoto et al. found that the degradation of cellulose and cellulose derivatives in canine specimens depended significantly on the cellulose crystalline form and chemical derivatization. Regenerated cellulose prepared by deacetylation of cellulose acetate (presumably the highly crystalline cellulose II polymorph) did not measurably degrade over the course of the 6-week experiment. By contrast, however, up to 75% (w/w) of equivalent samples of amorphous regenerated cellulose were degraded and absorbed over the same experimental period. Even greater degradation and absorption was observed for the methyl derivative while no degradation was observed for the ethyl derivative. In terms of biocompatibility, histological assessments of foreign body responses showed that all cellulosic samples were relatively well tolerated; although only the highly crystalline regenerated cellulose induced no immunological response.^[5]

Some years later, a long-term 60-week implantation study was carried out by Märtson et al. to determine the biocompatibility of a composite cellulose sponge consisting of cotton fibers surrounded by a porous matrix of cellulose regenerated from a viscose solution (sodium xantogenate). Histological assessment of the subcutaneously implanted samples revealed that the porous structures had become filled with connective tissue after just 4 weeks. Although the implants did trigger a moderate foreign body response, this was observed to diminish over the course of the study. Degradation of the implants was observed from approximately 16 weeks, with softening of the pore walls and fragmentation of the implant. The implants were not fully degraded over the course of the 60 weeks, however, leading the authors to designate the composite sponge, and cellulose generally as "a slowly degradable implantation material".^[6]

More recently, Yokota et al. produced model nanofilms of different cellulose polymorphs and derivatives in an elegant attempt to assess the effects of cellulose crystal formation and derivatization on bioactivity in vitro. A cellulose solution in N-methylmorpholine-N-oxide was modified at the reducing end with thiosemicarbazide. Thin films of cellulose II were then cast by spin coating onto glass followed by coagulation in ethanol. Self-assembled monolayers (SAMs) were also prepared from the cellulose-thiosemicarbazide N-methylmorpholine N-oxide solution on gold-sputtered surfaces, thereby effectively forcing a cellulose I crystal structure upon the regenerated cellulose by assembling the cellulose reducing ends and ensuring a parallel arrangement of polymer chains. Similar SAMs were also prepared for methylcellulose and hydroxyethylcellulose. Rat hepatocytes were cultured on the various surfaces with strikingly different results. Almost no cells adhered to the cellulose II or hydroxyethylcellulose surfaces, whereas the cells adhered and proliferated well on the cellulose I SAMs and methylcellulose surfaces. Indeed, some attachment was even observed on the cellulose I SAMs in serum-free conditions, suggesting that specific interactions may exist between these surfaces and rat hepatocytes.^[7]

In summary, the different studies assessing the biocompatibility and degradation of cellulose are difficult to compare due to the range of methodologies and cellulose preparations. In general, however, cellulose may be considered to be broadly biocompatible, invoking only moderate (if any) foreign body responses in vivo. In terms of degradation, cellulose may be considered nonbiodegradable in vivo or, at best, slowly degradable, due to the lack of cellulase enzymes in animals. However, the form (i.e., crystallinity, hydration and swelling) of the cellulose may affect the degree of degradation, absorption and immune response observed. It should also be noted that the toxicity of nanofibrillar or nanoparticulate materials depends, at least partially, on the morphology of the particles.^[8] With respect to nanofibrillar cellulosic materials it is, therefore, sensible to judge the biocompatibility of such materials on a case-by-case basis, although purely on a bulk (chemical) basis, cellulose may be considered broadly biocompatible.

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Executive Summary

Background

Cellulose is the principle structural polysaccharide in plants, but is also produced by certain strains of bacteria such as Gluconacetobacter xylinum.
Cellulose is broadly biocompatible, triggering limited (if any) immune response in vivo. The degree of biocompatibility both in vitro and in vivo is dependent upon the crystal structure and form of the cellulose.
Cellulose is nonbiodegradable in vivo, but under certain circumstances may be regarded as 'slowly degradable'.

Bacterial Cellulose

Bacterial cellulose (BC) from G. xylinum is produced as a porous nanofibrillar structure consisting of fibrils with diameters of less than 100 nm. The 'pellicles' produced under static culture may contain up to 99% water.

BC for Tissue Engineering

The most progress has been made in the field of vascular tissue engineering, using tubular scaffolds of BC as artificial vascular grafts. Efficacy has been demonstrated in vivo in animal models with a promising degree of function.
In vitro proof-of-principle studies, as well as initial in vivo studies, have also been carried out for bone, cartilage and nerve tissue engineering. A single study has also been carried out on the use of BC for engineering urinary conduits.
Several methods have been published for the modification of BC for improved bioactive properties. Scaffolds with multiscale porosity have been developed as well as composites with hydroxyapatite.
Novel constructs have employed cellulose-binding motifs from xyloglucan or cellulose-binding domains in order to decorate BC with specific bioactive peptide sequences. Such methods may improve the bioactivity of BC for tissue engineering.

Cellulose Nanowhiskers

Cellulose nanowhiskers (CNWs) are colloidal rod-like nanoparticles prepared by partial hydrolysis of native cellulose.
CNWs are approximately 5–20 nm in diameter by 150–1000 nm in length and are, therefore, several orders of magnitude smaller than animal cells.

Biomedical Applications for CNWs

Several studies have demonstrated that colloidal suspensions of CNWs may be slightly cytotoxic, but may also cross the plasma membrane of cells, pointing to applications in targeted drug delivery and bioimaging.
The cytotoxicity and ability to cross the plasma membrane appear to be dependent upon surface modification (e.g., surface charge).
CNWs may provide nanoscale structural cues to direct cells in tissue engineering applications.

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