1. Introduction
In recent years, there has been significant progress in biotechnology, and its increasing application in medicine can be observed. Biotechnology allows for improving results in many medical fields, including surgery. This is particularly noticeable in the field of reconstructive surgery, where the most important problem is the tissue deficit resulting from the defect usually caused by the surgery itself. Doctors for centuries and biotechnologists since the establishment of this discipline, i.e., from the beginning of the 1970s, have been trying to obtain materials to replace lost human tissues as a result of injuries, diseases, and underdevelopment due to congenital defects. A surgical specialty that deals with the repair of body deformities resulting from the above-mentioned causes is plastic and reconstructive surgery. A frequent problem is the lack or shortage of soft tissues of the body’s integumentary system caused by a congenital defect, trauma, or, more and more often, cancer. That is why it is important to find material that allows for easy reconstruction of natural tissues without local inflammatory reaction, which is common when in contact with most foreign materials. The basic method of reconstruction is harvesting the patient’s own tissues (autologous reconstruction methods), but it is often difficult or impossible—e.g., due to the excessive amount of tissue necessary for reconstruction to be successful and functional, or insufficient tissue quality. Autologous reconstructions are also often associated with a large wound surface resulting from the creation of a donor site from which tissues are taken. This can lead to poorer healing and often longer hospitalization, which is not expected or taken well by the patients. Moreover, the transfer of tissue flaps is usually associated with the risk of local ischemia and sometimes ischemia of the whole flap, which carries the risk of local or systemic infection. The risk of complications due to ischemia and tissue harvesting from the donor site also leads to larger scars, which reduces the final aesthetic satisfaction of the patient which is often expected when patients are treated by a plastic surgeon. Therefore, it is desirable to supplement the cavities with entirely artificial materials (implants) or materials of natural origin (biotechnological products). To fulfill their function, these materials must have several features, such as no or minimal immune reaction, good integration with host tissues, ease of modeling, mechanical strength, easy availability, and low cost [
1]. The implants used so far may contain metals, organometallic compounds, polypropylene and polyethylene derivatives, or silicone gel compounds. All of these could trigger a local or systemic immune response. At the same time, the knowledge of biomaterials is expanding, among which one of the most interesting and promising is bacterial cellulose (BC) produced by the
Komagataeibacter xylinum E25 strain [
2]. Bacterial cellulose (BC) is a linear polysaccharide composed of β-d-glucopyranose monomers linked by β-1,4-glycosidic linkages. It is an exopolysaccharide free of endotoxins and composed only of biocellulose fibers. The biochemical reactions of BC biosynthesis by
K. xylinus are well characterized. This is a precise and specifically regulated advanced process, involving many individual enzymes and complexes of catalytic and regulatory proteins. BC is a flexible membrane with high crystallinity, mechanical strength, a high degree of hydration, and thermal stability. It is characterized by a significant possibility of shaping the surface [
3]. BC is biocompatible, hypoallergenic, non-mutagenic, and non-teratogenic, similar to the collagen found in the human body. These properties enable BC to be used in regenerative and reconstructive medicine. In addition, its use avoids several problems. These include problems of finding a donor (in the case of allogeneic transplants), damaging the donor site, and creating a wound and excessive scarring (in the case of autogenic transplants), as well as problems with the rejection of transplants [
2]. So far, BC has been used to produce dressings with proven clinical effectiveness in human studies and structures that replace or strengthen anatomical structures such as large blood vessels, heart valves, fasciae, or trachea in animal studies [
3,
4,
5,
6]. The efficient production of bacterial cellulose meeting the requirements of biomedicine is the subject of many studies summarized in review articles and patents [
3]. However, few of them concern experimental work on animals. Considering the above-mentioned features of BC, the aim of the present study was a clinical and histopathological evaluation of a membrane made of bacterial cellulose used to fill defects in the auricle and rectus muscle in a domestic pig animal model. Muscle and cartilage defects are common problems in reconstructive surgery, both in the face and body. Craniofacial injuries very often involve protruding elements, including the nose and ears, which are associated with damage to the cartilaginous parts that constitute the frame. Defects resulting from chronic wounds or resections for oncological reasons are usually associated with large muscle losses. The closure of such defects requires various muscle flaps to be functional. Therefore, the study of a material that would be suitable as a replacement for these tissues is extremely important.
3. Results
3.1. Clinical Evaluation
During the 3-month observation of the animals, no clinical signs of local or generalized inflammatory reaction were found. The animals were in good general condition throughout the observation period, and there was no fever or signs of generalized infection. There were no signs of improper wound healing in the places where the implant was placed. Clinically, both in the skin of the operated ear and in the skin of the lower abdomen after hair removal, it was difficult to find the scar lines. Within the ear, the surgical sites could be palpated as a thickening on the skin, while the elasticity of the cartilages was maintained. There were no visible signs of surgical intervention in the form of scars within the skin of the lower abdomen, and the implantation site was recognized after comparing it with intraoperative pictures of the implantation site. By touch, the skin of the lower abdomen was smooth, maintaining its natural shape and consistency (
Figure 2). There were no signs of inflammation in the form of redness, swelling, or exudate in the places of implantation (
Figure 2).
3.2. Histopathological Evaluation
Among the analyzed materials, in one case (animal no. 1) of the abdominal integuments, it was found that the implant was partially resorbed and suppurated on a microscopic level, demarcated by fibrosis. In the other two, the implants were completely resorbed and fibrous scar tissue was formed. Single focal granulomas were integrated into the surroundings of the scar and could have been reactions to sutures. In one ear implant (animal no. 3), the changes were similar to those of animal no. 1 in the abdominal tissues, i.e., a fragment of the implant surrounded by a histiocytic reaction was preserved, but without a granulocytic infiltration (no suppuration). In other cases, the effect of the process was the formation of scars, among which there was visible granulomatous inflammation of the interstitial type (Panels B–D in
Figure 3).
In two of the analyzed cases, the presence of preserved cellulose was found (in one ear and in one case with the coatings). In one case (muscle, pig no. 2), the excised site did not contain any signs of BC or reaction to BC, and thus it was deemed as a “missed excision site”. Details of histopathological evaluation are presented in
Table 1.
In pig no. 1, in the ear location, congestion was assessed as mild, chronic inflammation was assessed as medium, there was no purulent infiltration, fibrosis was of a medium degree, giant cells were present, and cellulose was absent.
In pig no. 2, in the ear location, congestion was assessed as mild, chronic inflammation was assessed as medium, there was no purulent infiltration, fibrosis was of a medium degree, giant cells were absent, and cellulose was also absent.
In pig no. 3, in the ear location, congestion was assessed as mild, chronic inflammation was assessed as high, there was no purulent infiltration, fibrosis was of a medium degree, and giant cells and cellulose were present.
In pig no. 1, in the muscle location, congestion was assessed as medium, chronic inflammation was assessed as high, purulent infiltration was present, fibrosis was of a medium degree, and giant cells and cellulose were present.
In pig no. 2, in the muscle location, the excision site was missed and the histopathological evaluation showed no signs of BC implantation.
In pig no. 3, in the muscle location, there was no congestion, chronic inflammation was assessed as slight, purulent infiltration was absent, fibrosis was of a medium degree, and giant cells and cellulose were absent.
4. Discussion
Modern surgery requires minimal damage to the body’s integuments in the treatment process. Reconstructive surgery using foreign materials aims to meet these requirements as much as possible. The literature describes many materials to fill in the resulting tissue defects. The basic method is to use the patient’s own tissues, but there are products such as AlloDerm, polyurethane, and polyethylene that are well researched and their use is well documented [
11,
12,
13]. The usefulness of bacterial cellulose (BC) is very promising; however, its use is not yet well established and researched. The lack of the cellulase enzyme in animals means that it should not be broken down within their tissues. In humans, there is also no known mechanism for its degradation. This feature, as well as many others (e.g., the possibility to change the structure or shape), makes it possible to modify its application. Currently, the most advanced research is carried out on the use of BC in cardiac surgery to use it as heart valves [
5] and in vascular surgery as substitutes for blood vessel walls [
14]. Research assessing BC in reconstructive surgery is still scarce, which is partially bridged by the current work [
15,
16].
The method of producing bacterial cellulose is simple and relatively cheap, which makes it competitive with other implantable materials. The cellulose itself, as proved in previous studies, is indifferent to the host organism, does not cause rejection reactions, and should not be biodegradable. The bacterial cellulose used in the study was checked in terms of mechanical and physical conditions prior to implantation in the animal, as shown in previous studies [
14].
There are a number of different methods for assessing implant biocompatibility in a donor found in the literature. It is possible to evaluate the graft by histological studies [
14,
17], biochemical studies [
18], tissue and cell cultures [
19], histochemical analyses [
20], and perfusion studies of the whole organ [
21]. Other physical features such as weight, elasticity, stiffness, mechanical malfunctions, elongation, and surface damage are possible to observe using electron microscopy [
22]. Moreover, there are also newer technical possibilities utilizing radiology. X-ray [
23], magnetic resonance imaging [
24], and computed tomography [
25] both with or without radiological contrast or radioactive isotopes have been helpful in observing possible inflammatory responses that could occur when using metallic implants and be caused by their decomposition and contamination of the operating site or even circulation in the bloodstream of a patient. Due to the high costs of most of the above-mentioned methods, the most commonly used method in experimental studies is a histological assessment with the use of hematoxylin–eosin dye.
In this study, clinical and histopathological examinations were used to evaluate the behavior of BC in living tissues. The assessed parts of pig ears and abdominal covers had an aesthetic appearance that did not differ from the normal ones, and the recipient sites did not show any signs of inflammation. The ear with the BC implant retained its stiffness and elasticity. Available cell-free matrices do not achieve the stiffness that can be obtained with BC, and therefore can be compared with artificial materials. In their case, however, there is a frequent risk of necrosis of living tissues in the area and the need to remove the implanted material. In the area of the ear, two out of three cases were resorbed with the formation of fibrous tissue, and in one case, there was no resorption. There were no signs of contamination with the features of rejection of the grafted material, which is often the case with typical artificial materials, such as polypropylene. These observations are confirmed in the work of Amorim et al., who studied the behavior of bacterial cellulose filling the defects of the nasal septum in rabbits. Although the bacterial strain used in their work was different, it also did not cause rejection, with a satisfactory degree of integration with the animal’s tissues14.
Research on the biocompatibility of implantable materials usually requires evaluation at different times, considering tissue responses to foreign bodies. The time of the progression of the changes was important for the assessment of the local inflammation and the appearance of the ear after the implantation of the cellulose body during the observation process. No published studies on the use of such material in the reconstruction of auricle cartilage have been found in the literature. Possible further studies on a larger group of animals with longer observation times should provide additional information about the breakdown of biocellulose observed in some samples and its influence on the shape of the ear. In the present study, it could only be noticed that the biocellulose shaped body was suitably plastic and could be easily handled. It is easy to place under the skin, providing a natural substitute and consistency compared to the rest of the cartilage parts of the ear. Thus, in the future, BC may be a satisfactory substitute for the missing fragments of cartilage parts of the auricle, and maybe even the septum or wings of the nose. Within the abdominal wall, there were no signs of surface defects, cavities resulting from the lack of tissues, or scar contractures. In the histopathological examination, one out of three implants placed within the rectus abdominis muscles was completely resorbed, producing fibrous tissue in this place, and another one was partially resorbed, also with the formation of fibrous tissue.
The performed histological examination showed a tendency for the BC implant to be incorporated into the scar tissue. It is properly accepted by the surrounding tissues of the animal without the excessive or pathological inflammatory response typical of a foreign body. The scar formed after implantation of the BC effectively joined the edges of the cavities and created a barrier separating healthy tissues from other artificial materials used in similar situations. We consider this moderate amount of scarred fibrous tissue to be beneficial, because mechanically, it is not a fully valuable but overall beneficial substitute for the tissue. This is also indicated as an advantage of BC material by other authors.
Important consideration arising from the present study is the biodegradability of BC, as this material, as mentioned, theoretically should not undergo biodegradation. However, as shown by the results of the present study, BC was found only in two of five excision sites upon histopathological examination. It is important to underline that the primary aim of BC use in reconstructive surgery is its clinical usefulness—specifically, the mechanical and aesthetic properties of the body site where BC was implanted. As shown in this study, these aims (proper filling of a defect) were properly achieved regardless of the result of histopathological examination. Therefore, while more research is needed to explain the course of BC degradation in mammals (as it can be expected that human subjects will show similar breakdown mechanisms as Sus scorfa domesticus), we can already draw conclusions that it is an adequate material for reconstructive properties.
There are also important considerations of this study. Primarily, this study included only three subjects with two sites each—six sites total. It could be desired to increase that number, but considering the ethics of animal studies, costs, and the consistent and promising results of the present study, the authors do not feel that it is needed to increase the number of subjects or to repeat the experiments. Further, this study did not explain the mechanism of BC degradation, but this process was observed. This warrants further studies to explain the specifics of this phenomenon. Finally, the main strengths of this study are the multidisciplinary team and the translational character of the study. As a group of various researchers— biomaterials experts, veterinarians, and plastic surgeons—we have made an important step toward the routine use of BC in plastic and reconstructive surgery.