The purpose of this study was to evaluate the morphological characteristics of the reparative processes in the soft tissues of the lower extremities in patients with neuropathic and neuroischaemic forms of DFS.

The study included 63 patients (21 women and 42 men) with neuropathic and neuroischaemic forms (after revascularisation and without critical ischaemia) of DFS who underwent surgical treatment of the wound defect. After the surgical treatment, tissue samples were taken for biopsy, followed by histological and immunohistochemical (IHC) studies. Table 1 shows the clinical characteristics of the examined patients.

Table 1. Clinical characteristics and laboratory test results of patients

IQR, interquartile range; DM, diabetes mellitus; HbA1c, glycated haemoglobin; HDL, high-density lipoprotein; LDL, low-density lipoprotein; ESR, erythrocyte sedimentation rate; GFR, glomerular filtration rate.

All laboratory tests were performed according to standard methods of diagnostic laboratories at Endocrinology Research Centre, Moscow, Russian Federation (Ilyin A.V., Head of department).

The area of the wound was determined by delineating the contours of the wound through the transparent film (Opsite Flexigrid, Smith & Nephew), followed by a calculation of the area inside the contour. For transcutaneous oximetry, Radiometer (Denmark) equipment was used. Sensors were positioned at a distance of 0.5–1 cm from the edges of the wound. Morphological studies were performed at the Laboratory of Histology and Immunohistochemistry jointly with Criobank group FGBI Endocrinology Research Center (Department Head prof. A.J. Abrosimov). Biopsy material was analysed using histological and IHC techniques. Biopsy samples (0.5 mL) were taken from the central area of the wound bed. The material was fixed in 10% formalin solution and then embedded in paraffin in the usual manner. Serial sections (3–5 μm thick) were deparaffinised in the standard way and stained with hematoxylin and eosin. Histological evaluation of the severity of oedema and ECM was performed using semi-quantitative scoring as follows: +, poorly expressed; ++, moderately expressed; +++, strongly expressed; and ++++, very strongly expressed.

Granulation tissue maturity was evaluated by determining the area of necrosis, number of blood vessels, severity of inflammatory infiltrate and cell composition (polymorphonuclear leucocytes, lymphocytes, macrophages and fibroblasts). Granulation tissue development was subdivided into three types: young, intermediate, and mature. Young granulation tissue is rich in cells and thin-walled blood vessels in the connective tissue, with a large number of lymphocyte-like, undifferentiated connective tissue cells, leucocytes, plasma cells and mast cells between blood vessels. In intermediate (transitional) granulation tissue, differentiation of cells, fibrous structures and blood vessels continues. The number of haematogenous cells decreases, and the number of fibroblasts increases. Because of the production of collagen by fibroblasts, first argyrophilic and then collagen fibrils develop in the intercellular space. As the fibroblasts mature, the amount of collagen fibrils increases and they form collagen fibres. Mature granulation tissue is fibrous connective tissue.

The phases of inflammation were determined according to Kuzin (1990), with phase 1 characterized by a predominance of neutrophilic granulocytes, monocytes and macrophages; phase 2 characterised by regeneration and maturation of the granulation tissue, which is formed in separate pockets on the bottom of the wound and characterised by intense capillary neovascularisation, and large numbers of fibroblasts and development of collagen and elastin fibres; and phase 3 characterised by scar development and epithelisation, with granulation tissue being converted into a mature fibrous tissue with fibrocytes and coarse collagen fibres.

For IHC studies, the slides were processed on a Leica BOND-MAX immunostainer. Antibodies to TIMP-1, MMP-9, macrophage marker CD68 and osteopontin (Dako, Glostrup, Denmark) were used in standard dilutions and with positive controls. Expression of cytoplasmic markers (TIMP-1, MMP-9 and osteopontin) was assessed semi-quantitatively: +, only individual cells stained (<30% of the cells); ++, 30%–60% of cells stained; +++, 60%–90% of cells stained and ++++, >90% of cells stained.

The degree of expression of CD68 (a marker of macrophages and myeloid histiocytic cells) was estimated semi-quantitatively from the total number of immunopositive cells and varied in the following ranges: 5%–10%, 10%–15%, 15%–20%, 20%–30% and 30%–40% of cells stained with specific antibodies. Photomicrographs were taken with a DFC450 C camera (Leica, Germany).

The study protocol was approved by the Ethics Committee of Endocrinology Research Centre (28 November 2012; Minutes No. 18). All patients signed an informed consent.

Statistical analysis was performed using Statistica software version 7.0 (StatSoft). Statistical parameters used for analysis were median, interquartile range and Spearman’s rank correlation coefficient.

The paper presents for the first time a comprehensive assessment of the level of intensity of repair processes in the soft tissues of the lower limbs in patients with DFS using chosen morphological techniques.

Biopsy material was taken from the bottom of the wound defects after surgery but before local therapy treatment during the study. The biopsy material was subjected to histological and histochemical studies.

All wounds were characterised by marked intercellular oedema with a median severity of 3 (3, 4); poorly organised ECM with a median value of 2 (1, 2); low content of fibroblast-like cells and significant inflammatory infiltration and the presence of young granulation tissue with a median value of 1 (1; 2). Despite the fact that in many patients wound healing process was protracted and resistant to local treatment, surgical treatment allowed a transformation from a chronic to an acute process. The majority of patients studied had wounds in inflammation phase with a median value of 1 (1, 2). Figure 1 shows a typical histological picture of the biopsy material from a wound defect before local treatment.

IHC features of wound healing process in patients with DFS

IHC studies demonstrated the presence of a moderate amount of macrophages (immunopositive to CD68 antibodies), intensive staining for MMP-9 and weak staining for TIMP-1 and osteopontin in all patients (Table 2).

Table 2. Expression of repair process markers according to immunohistochemical studies

IQR, interquartile range; OPN, osteopontin; MMP matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases.

Despite the long history of diabetes in the examined patients, there was no correlation (Rs = -0.01) between the duration of the disease, the value of glycated haemoglobin (HbA1c) and the intensity of the reparative processes in the soft tissues of the lower limbs.

Figures 2–5 show the results of IHC studies of wound biopsies after the surgical treatment.

Chronic wound defects of the lower extremities in diabetic patients are becoming increasingly common due, in particular, to the growing incidence of DM. Healing processes are slowed down when carbohydrate metabolism is impaired, but the pathophysiological basis of this fact is not fully understood. The purpose of this study was to evaluate the morphological and IHC repair indicators in patients with DM.

This paper presents a comprehensive study of 63 patients with DFS, aged 32–78 years old, who were admitted to the Department of the Diabetic Foot at FGBI Endocrinology Center, Ministry of Health, Russian Federation, between September 2012 and December 2014. All patients underwent surgical treatment of the wound defect, antibacterial therapy and reduction of load on the affected limb. During in-patient treatment, all patients underwent correction of dietary therapy with the goal of achieving individual targets for glycaemia indicators [17].

Clinical examination and laboratory test results showed that the median HbAqc levels in all patients was 8.7% (7.6, 10); thus, the carbohydrate metabolism of the patients was unsatisfactory. Christman et al. [18] demonstrated that the rate of lower-extremity wound healing in patients with diabetes is directly correlated to the level of HbA1c. With HbA1c ≤7%, the healing rate was 0.197 cm2/day; if HbA1c was 7%–8%, wound defects decreased in size at the rate 0.157 cm2/day; in patients with HbA1c ≥8% the rate of healing was 0.028 cm2/day.

Most patients did not have diabetic nephropathy or were at the stage of microalbuminuria, which means that protein deficiency as a factor unfavourable for tissue healing was absent, which was confirmed by laboratory tests.

The majority of patients had diabetic polyneuropathy. Despite the fact that in a small number of patients, there was no decrease of peripheral sensitivity, hyperkeratosis and dyshidrosis were present, which allowed us to interpret the above symptoms as the initial manifestation of a reduction of the peripheral innervation.

The histological examination of wound defect biopsy samples taken before local treatment showed that all patients had oedema, poorly organised ECM, low content of fibroblast-like cells and a significant inflammatory infiltrate. All wounds were in the inflammation phase of healing. These findings are consistent with those of Boulton et al. who demonstrated that diabetic wounds have a long inflammatory phase, decreased activity of inflammatory cells and delayed ECM reorganisation. These disturbances prevent healing and foster the transition of the wound from the acute to the chronic form [6].

According to Baltziz [8], the interaction between glycated collagen, ECM and fibroblasts or endothelial cells prevents cell adhesion, proliferation and reduction of the wound defect size. IHC studies also demonstrated the slowing of reparative processes in the soft tissues of the lower extremities in patients with DM. In the present study, we found that the level of osteopontin expression was reduced in the majority of patients. This is consistent with the literature, which refers to the decrease in the expression of osteopontin in patients with impaired glucose metabolism [13]. It was suggested that one of the reasons for the reduced rate of reparative processes in patients with DM is a decrease in osteopontin expression in the wound bed, which manifests as deficient migration of immunocompetent cells, accumulation of decomposition products, decreased number of endothelial cells, slow neoangiogenesis and disorganisation of ECM [13].

Moderate-intensity staining for macrophage marker CD68 was noted in all patients. In experiments on laboratory models, it was established that in the presence of DM, it takes longer for macrophages to accumulate in the wound, which may be associated with increased levels of pro-inflammatory cytokines and proteases and reduced levels of growth factors. Similar results were obtained in patients with diabetes [8].

The increased MMP-9 expression and weak staining of TIMP-1 observed following surgical treatment indicate high proteolytic activity in wounds, and according to some studies, result in slower collagen reorganisation and is predictive of poor prognosis in wound healing. For example, Liu et al. [15] noted that high MMP-9 levels in the wound fluid is indicative of inflammation and is a marker of poor wound healing in patients with DM. Other researchers suggested using the ratio of MMP-9/TIMP-1 as a predictor of wound healing in patients with DFS [16]. Although there was no control group in the present study, our results are consistent with published data.

Thus, on the basis of these data, we can assert that in patients with DM, reparative processes in soft tissues are slowed down, but currently, the amount of data on the long-term results of treatment is insufficient, and further research is required.

The study was conducted under the sponsorship of FGBI Endocrinology Research Centre within the approved program of research.

The authors declare the absence of explicit and potential conflicts of interest associated with the publication of this paper.

The contribution of each author: E.L. Zaitseva — collection and processing of material, data analysis and writing of the text; Doronina L.P. — collection of material; Molchkov R.V. — a histological study; Voronkova I.A. — immunohistochemical studies; Tokmakova A.Y. — the concept and design of the study.