## 2 Materials and Methods
### Morphological Studies and Immunohistochemistryistochemistry  
Tissue biopsy samples from the edges and bottom of bedsores (1 sample from each zone) were fixed in 10% formalin solution and then embedded in paraffin. Sections 3-4 \(\upmu\)m thick were stained with hematoxylin and eosin before viewing. The sample size was 6 (15.7%) observations in the main group and 4 (13.7%) in the control group. Histological examination in each observation was performed three times--before the start of treatment, on the 14 th and 28 th days from the start of the procedure for activating preparative processes in the DU zone. For IHC studies, serial paraffin sections 4 \(\upmu\)m thick were made, located on slides coated with a polylysine layer (Menzel Glaser Polylisine, Germany). Unstained sections were processed using a standard method of immunohistochemistry with thermal unmasking of antigens. Demasking was carried out in a water bath with a PT Module microprocessor (Thermo Scientific, Waltham, MA, USA); polylysine slides with paraffin sections were deparaffinized according to the standard method and, after rinsing in distilled water, were immersed in containers with a universal buffer for additional deparaffinization, unmasking, and rehydration of Trilogy sections (Cell Marque, Rocklin, CA, USA) and heated in a water bath to 95 \({}^{\circ}\)C for 20 min. The slides were then cooled at room temperature for 20 min. All further stages of the immunohistochemical reaction to prevent drying of the sections were carried out in a humid chamber SlideMaster (Bio Optica, Milano, Italy). To block endogenous peroxidase, slides were incubated for 10 min with a peroxidase inhibitor, after which the sections were rinsed in phosphate buffer (pH 7.0-7.6) (Cell Marque, USA) and incubated with Ultra-V-Block to block nonspecific protein interactions (LabVision, Fremont, CA, USA) within 30 min. At the end of incubation, excess reagent was gently shaken off the slides and primary antibodies were applied. USA) and incubated with Ultra-V-Block (LabVision, USA) for 30 min to block nonspecific protein interactions. Mouse monoclonal antibodies to type I collagen (clone 3G3, Santa Cruz, Dallas, TX, USA, 1:100 dilution), type III collagen (clone B-4, Santa Cruz, 1:50 dilution), and type IV collagen (clone COL-94, Santa Cruz, dilution 1:50), MMP-1 (clone 3B6, Santa Cruz, dilution 1:100), SMA (clone 1A4, Dako Agilent, Santa Clara CA, USA, dilution 1:100) and TGF\(\beta\) (clone 3C11, Santa Cruz, dilution 1:100). Sections were incubated with primary antibodies for 30 min according to the manufacturer's specification for the antibody. After incubation, the sections were thoroughly washed in phosphate buffer (pH 7.0-7.6) to remove primary antibodies that did not bind to epitopes. To detect primary antibodies bound to the corresponding antigens, the Histofine(r) Simple Stain MAX PO (MULTI) universal polymer system (Histofine, Nichirei, Japan) was used, containing a dextran framework with multiply attached molecules of the horseradishish peroxidase enzyme and secondary antibodies to antimouse and antirabbit immunoglobulins (Ig). The time of incubation of sections with the polymer detection system in a humid chamber was 30 min. At the end of the incubation, the sections were rinsed in phosphate buffer (pH 7.0-7.6). Depending on the required (desired) intensity of staining, the sections were incubated with DAB for 5-10 min. Next, the slides were washed in distilled water and the nuclei were stained with Mayer's hematoxylin for 2-3 min. After that, the slides were dehydrated in a battery consisting of distilled water, alcohols of increasing concentration (70%, 80%, and 95% absolute alcohol) and 3 xylenes. After that, the sections were covered with coverslips using BioMount synthetic medium (Bio Optica, Italy). When setting up immunohistochemical reactions, positive and negative controls were used. As negative controls, samples of the studied sections were taken, which were subjected to the standard IHC study procedure, but without incubation with primary antibodies.