Biological Evidence of Improved Wound Healing Using Autologous Micrografts in a Diabetic Animal Model

Abstract

Background: Tissue healing consists of four main phases: coagulation, inflammation, proliferation, and remodeling. In diabetic patients, this process is stagnant in the inflammatory stage, leading to chronic wounds. The aim of this study is to evaluate in an animal model the biological evidence related to the use of the Rigenera^® technology (Turin Italy), an innovative mechanical procedure to isolate autologous micrografts (AMG). Methods: Fifty male Wistar rats were divided into four groups: control (C), control treated with micrografts (CM), diabetic (DB), and diabetic treated with micrografts (DBM). The experimental setup involved: the quantification of the total collagen and elastic fibers; histopathological analysis; immunohistochemical analysis for collagen type I (COL1), collagen type III (COL3), vascular endothelial growth factor (VEGF-A), and interleukin 4 (IL4) and 10 (IL10); evaluation of the oxidative stress; measurement of gluthatione (GSH); and, finally, an enzyme-linked immunosorbent assay (ELISA) on tumor necrosis factor-α (TNF-α). Results: The AMG technology induces a faster healing process: VEGF-A, IL4, IL10, and GSH increased, while TNF-α and oxidative stress decreased. Conclusions: Animals treated with micrografts showed more favorable results for healing compared to those that did not receive treatment, demonstrating a positive participation of the micrografts in the treatment of difficult-to-heal wounds.

1. Introduction

The skin is considered the largest organ in the human body. In an adult, it represents about 16% of the total body weight, and, when distended, it occupies an area of approximately two square meters. Histologically, it is divided into the epidermis, dermis, and hypodermis (also known as the subcutaneous fat layer) [1]. The epidermis is the superficial layer of the skin, and it is composed of keratinized, stratified, and squamous epithelial tissue (Figure 1). It is avascular and receives nutrients by diffusion through the layer just below it, the dermis. In the epidermis, there are distinct layers arranged from the outermost to the innermost region, including the stratum corneum, stratum lucida, stratum granulosum, and stratum spinosum [2]. The deepest layer, known as the basal layer, is situated adjacent to the dermis, with finger-like projections towards it, referred to as the dermal papillae. The presence of these projections, known as rete ridges, is absent in the early stages of wound healing, which renders the wound more vulnerable to injury if exposed to trauma [3].

The basal layer is formed by a single row of cuboidal or columnar keratinocytes, interspersed with stem cells responsible for the renewal of resident cells; it has a great ability to proliferate, and daughter cells migrate towards the skin surface. That suprabasal migration is accompanied by cell maturation until the formation of corneocytes on the skin surface [4]. Their cytoskeleton contains intermediate filaments formed by keratin that attach to desmosomes. In this way, the cells of the basal layer attach to each other and to the adjacent spinous layer [5]. When attached to hemidesmosomes, actin filaments allow the attachment of keratinocytes to the basement membrane, located between the epidermis and the dermis [6].

There are four main types of cells in the epidermis: keratinocytes, melanocytes, Langerhans cells, and Merkel cells. Keratinocytes make up about 90% of the epidermis, and the source of replacement of these cells is the basal layer; they produce the keratin protein that provides protection to the skin and the underlying tissues against heat, chemical agents, and microorganisms [7].

Keratinocytes are identified as the primary cell responsible for the healing of wounds of the epidermis and for maintaining tissue integrity. A stratified keratinized epithelium undergoes constant turnover, regenerating completely in 48 h. Keratinocytes originating from the basal layer are devoid of keratin and begin to accumulate more and more of this protein as they pass from one epidermal layer to another. This process is accelerated in the case of injuries, such as abrasions and burns [8].

Melanocytes are cells with long and thin processes, called dendrites, placed between the keratinocytes. They correspond to 8% of skin cells and release melanin pigment granules that are absorbed by the keratinocytes. Once in keratinocytes, melanin forms a barrier around the core on the side facing the skin surface. In this way, melanin protects cellular DNA against the deleterious effects of ultraviolet radiation [9].

The Langerhans cells migrate to the epidermis from the bone marrow. They represent a small percentage of the total epidermis cells and participate in the immune response.

Merkel cells are located deeper in the epidermis, where they come into contact with sensory neurons; together with them, they participate in the sense of touch [10].

The dermis is the layer that lies just below the epidermis and receives the increased blood supply to the skin. Most skin appendages are in the dermis: apocrine glands, eccrine glands, and hair follicles. The dermis is a connective tissue containing collagen and elastic fibers. Fibroblasts, macrophages, and adipocytes constitute the cellular component from the dermis. It has two layers: superficial or papillary and deep dermis or reticulate [11].

The superficial layer corresponds to only one-fifth of the total dermis. It contains fine elastic fibers. Its total area is increased by the presence of dermal papillae, which are finger-like projections towards the epidermis. In some dermal papillae, corpuscles of Meissner (touch-sensitive nerve endings) and also free nerves (responsible for sensations of heat, cold, pain, tickling, and itching) are present [12].

In the deepest layer of the dermis, the reticular region, tissue is observed as dense, irregular connective tissue, with bundles of collagen fibers and thicker elastic fibers. Among the bundles of collagen fibers, follicles, hair, sweat, and sebaceous glands, nerves and fat cells can be observed. The combination of collagen fibers and elastic fibers gives the dermis the properties of elasticity, strength, and extensibility. When tissue damage reaches the reticular dermis, grafts are usually necessary [13].

The deepest layer of the skin is called the hypodermis or subcutaneous layer. It contains a large amount of adipose tissue, sensitive receptors pressure (Pacinian corpuscles), and large blood vessels that supply the dermis. Fibers that come from the dermis cross the hypodermis and attach to tissues and underlying organs [14].

The skin possesses an excellent regenerative capacity due to the presence of various mesenchymal stem cells (MSCs) located in its appendages (hair follicles, sebaceous glands, and sweat glands) as well as in the basal layer [15]. These cells exhibit a high degree of plasticity and are arranged in distinct compartments known as niches, where they interact with neighboring cells and a specific extracellular matrix to determine their functions [16,17,18]. In the skin, three different niches of MSC have been identified: (i) the basal layer of the epidermis, (ii) the hair follicle bulb (present in mice, but not in humans), and (iii) the base of the sebaceous gland duct, which suggests the existence of a niche also in the duct of the sweat glands [19,20,21].

A wound is defined as damage to or discontinuity of the structural anatomy of the skin and the consequent loss of its normal functions. It can be a simple interruption of the epithelial integrity or it can be deeper, extending to the subcutaneous tissue, with damage to various structures, such as tendons, muscles, vessels, nerves, organs’ parenchyma, and bones [22].

Healing consists of the reestablishment of the continuity of the epidermis, so that the tissue that differentiates it acts as a physical, chemical, and bacterial barrier, which is one of the skin’s vital functions [1]. The healing process requires an integrated expression of several chemokines, cytokines, growth factors, and cell types that are present in the wound from tissue injury to the final healing events. Throughout this process, their expression varies temporally and quantitatively. They are produced by cells present at the wound site and act in paracrine and autocrine signaling [23].

Wound healing can happen by regeneration or through the reparation process. When regeneration occurs, the tissue for reconstruction is the same as that of the healthy tissue. This can be observed in the superficial epidermis, in the mucous membranes, or in fetal skin. In tissue repair, the wound is filled with fibrotic tissue and presents with scars [11,24].

Tissues have different healing times. Wounds can be classified as acute or chronic according to the way they establish and evolve. Acute wounds heal within the period expected and in a way that is hassle-free. Chronic wounds are those that do not fulfill the progression of the normal healing stages, and the damage established is not repaired in the expected order and time. Many factors can contribute to the interruption and consequent damage of normal healing: infection, tissue hypoxia, necrosis, and exudates. Excessive amounts of inflammatory cytokines can prolong one or more phases of the healing time, leading to wound chronicity [23].

After the injury has occurred, to reestablish the anatomical condition and function (that is, healing), the following steps are necessary in the affected tissues: coagulation and hemostasis, the inflammatory phase, the proliferative phase, and the remodeling phase. These phases are sequential, overlapping, and interdependent such that in different areas of the same wound, distinct phases of the healing process can be observed. Thus, deficiency in any of the healing stages determines the failure of the event [25,26,27] (Figure 2).

Diabetes mellitus (DM) is an important global health problem caused by changes in nutritional habits, sedentary lifestyle, overweight, population growth and aging, increasing urbanization, and the greater survival of diabetic patients. It has a cost of billions of annual dollars that impacts the health systems of all countries, but it is more representative in underdeveloped countries. Type 1 diabetes is the carrier of exogenous insulin, while type 2 diabetes is more common and accounts for 85–90% of cases. Diabetes is characterized by disturbances in the secretion and action of insulin and can be managed with food control and physical exercise. Other less common types of diabetes have been reported, such as genetic defects of beta cells and of the insulin, degenerative diseases of the pancreas, diabetes related to other endocrinopathies, and drug diabetes. Complications include cardiovascular and cerebrovascular diseases, retinopathy, nephropathy, and ulcers and skin conditions [28]. Diabetic individuals have difficulty healing wounds in the extremities, especially in the feet. Diabetic foot (DF) wounds are caused by neuropathy and vascular insufficiency, with 20% having previous arterial occlusive disease, 50% having peripheral neuropathy, and 80% having both conditions. Any trauma leading to the formation of skin ulcers predisposes patients to complications [29,30]. Treatments for DF ulcers aim to increase vascularity and to break down physiological barriers that prevent healing, but the success of treatment depends on several factors. Chronic wounds of diabetic feet have their healing interrupted in the inflammatory phase due to deficiencies in cells involved in the process, as well as of chemokines, cytokines, and growth factors.

A possible countermeasure to treat chronic wounds in diabetic patients is the use of adult stem cells for tissue maintenance and repair [31]. Among them, mesenchymal stem cells (MSCs) are present throughout the organism and in the perivascular region of adult tissues. They are multipotent and capable of forming ectoderm, mesoderm, and endoderm cells [32], and they release exosomes to stimulate tissue regeneration and to regulate the immune system [33,34]. However, an important limitation of using MSCs lies in the necessity to cultivate cells, which involves enzymatic manipulation, leading additionally to an increase in the time to obtain them and the financial cost [35,36].

Rigenera^® technology (Human Brain Wave, Turin, Italy) is now a currently available alternative technology for obtaining micrografts (namely progenitor cells (PCs) within their own extracellular matrix) involving disposable medical devices as mechanical disruptors of biological tissues [37,38,39,40,41,42,43,44,45]. It allows for obtaining micrografts in an autologous, homologous, and minimally invasive manner, and these can be used immediately without the need for culture cells. Scientific studies have shown that the cellular population obtained after the mechanical disaggregation is positive for mesenchymal stem cells, with markers identifying those cells as PCs, and they have shown a viability ranging between 70 and 90%. AMTs have the ability to act both through cellular differentiation and in a paracrine manner through the release of secretomas containing various molecules that will act on neighboring cells; they have the potential to stimulate multiple events at once, allowing interdependent phases to be resolved endogenously [46].

In this study, diabetic foot wounds were used as an experimental model in order to verify the performance of AMTs in the evolution of the healing process in skin wounds. Fifty male Wistar rats were involved and divided into four groups: “control (C)”; “control treated with micrografts (CM)”; “diabetic (DB)”; and “diabetic treated with micrografts (DBM)”.

2. Results

2.1. Histopathological Analysis

Research findings on hematoxylin-eosin-stained slides revealed an extensive inflammatory infiltration among the granulation tissue seen in all groups at the start of healing (3 days). Nevertheless, the number of neutrophils in the CM group is substantially smaller (Figure 3). The images were acquired from samples collected 3 days after the creation of the artificial wound.

Despite the progression of the healing process, inflammatory cells remained in the diabetes groups (DB and DBM) longer than in the non-diabetic groups (C and CM) (Figure 4).

According to histological studies, the CM group is in the healing phase after 3 days, which is equal to the C group after 7 days of recovery. At 7 days, the proliferation phase begins, leading to fibroblasts migration, angiogenesis, and the commencement of epithelialization (Figure 5).

The epidermal tissue was detected at 3 days in the treatment groups (Figure 6), indicating a faster epithelialization in the micrograft groups.

Angiogenesis occurs in all groups; however, it takes longer to begin in the diabetic group, with detection occurring after just 7 days. Active fibroblasts were likewise discovered only after 7 days of recovery. The CM group has much more fibroblasts and collagen fiber production than the C group. Although there are still disordered regions, collagen is thicker than in the control group.

Granulation tissue with many newly formed capillaries and fibroblasts is present in the DBM group in the middle of the extracellular matrix formed by the newly formed fibrillar collagen synthetized. By 14 days (Figure 7), there was a full region of healing in all groups, with no discontinuity of the epidermis, and the healing was in the final phase of proliferation and the beginning of remodeling. Every layer of the epidermis is visible, from the stratum basale to the stratum cornea; however, the epidermis is thinner and has fewer keratin layers in the diabetic group. The formation of epidermal attachments has begun in the groups treated with the micrograft, but it is still lacking in the untreated groups. Granulation tissue has already been totally replaced by collagen, although collagen remains fibrillar, with thin and disordered fibers, with an appearance of newly formed collagen in the diabetic group. The majority of fibroblasts have already been replaced by fibrocytes.

2.2. Collagen and Elastic Fibers Quantification

The animals in the C group had less collagen at 7 and 14 days of evolution than the DBM group, which was still at 3 days of healing. As a result, the DBM group produced more collagen in a shorter healing time than the C groups with longer healing times (Figure 8).

The morphometric analysis for elastic fibers quantification revealed no statistically significant differences between the groups (Figure 9 and Figure 10).

From the statistical analysis conducted to quantify collagen production and elastic fibers, it was found that there is a statistical difference between C (7 days) and C (14 days) compared to DBM (3 days) for collagen (Figure 10a). However, no statistically significant differences were observed among the groups for elastic fibers (Figure 10b).

2.3. Immunohistochemical Analysis for COL1, COL3, VEGF-A, IL4, and IL10

Throughout the 3-day assessment, there were no significant changes in collagen I labeling. At 7 days, the CM group had more COL1 marking than the C group. The animals in the DB and DBM groups had the lowest marking and did not distinguish from one another. At 14 days, the same behavior was seen (Figure 11a).

At the 3-day assessment, there were no significant changes in collagen III labeling. At 7 days, tissues in the C and CM groups had more marking in their fragments, followed by the animals in the DBM group. The animals in the DB group had the least amount of staining. The animals in the CM and DBM groups had more marking at 14 days than the C and DB groups, with the latter having the lowest average (Figure 11b).

At 3 days, there were significant variations in the evaluation of VEGF, with skin samples from the CM and DBM groups demonstrating higher marking than the DB and C groups, with the latter showing the lowest markup. There were no variations in marking between the experimental groups after 7 days. By 14 days, however, the animals in the CM and DBM groups had more markings than the C and DB groups, which did not differ from each other (Figure 11c).

We observed the same trend at the 3- and 7-day evaluations, characterized by increased marking in the skin pieces of the CM and DBM animals, whereas the C and DB groups showed less staining without significant differences. The animals in the CM group, on the other hand, had more marking at 14 days. The animals in the C and DBM groups did not vary from one another, but the DB had less marking (Figure 11d).

During 3 and 7 days, there was no significant change in the labeling of this cytokine. By 14 days, however, the animals in the CM and DBM groups had more marking. The animals in the C and DB groups had less marking but did not vary from one another (Figure 11e).

2.4. Evaluation of the Oxidative Stress—Measurement of Gluthatione (GSH)

Tissue assessment of GSH levels at three points in the groups’ studies revealed that the groups treated with the micrograft had greater levels than the diabetic group (Figure 12a). In terms of tissue concentrations of skin lipid peroxidation (TBARS) levels, the DB group showed a substantial rise, but the DBST group showed a decrease when compared to the diabetic and control groups (Figure 12b).

2.5. Evaluation of Tumor Necrosis Factor-α (TNF-α)

In terms of TNF-α dose, the control group (C) had a greater concentration than the other experimental groups on the third day. However, on the seventh and fourteenth days, the diabetic (DB) group had a greater concentration, contrasting with the other groups’ findings (Figure 13).

3. Discussion

The process of tissue healing involves a series of events that are interdependent and involve various chemokines, cytokines, and growth factors produced by cells that participate in tissue repair [11,23,24,25]. This process goes through phases of coagulation and hemostasis, inflammation, proliferation, and tissue remodeling [24]. In extremity wounds of diabetic individuals, the healing process is interrupted in the inflammatory phase due to a large amount of cytokines, chemokines, and pro-inflammatory factors that prevent progression to the proliferation phase and result in chronic wounds [47].

Stem cells are seen as a possible solution to this problem as they can release exosomes containing factors that positively interfere in healing and also differentiate into other cell types, which can act on stagnant healing and act on several factors simultaneously [48].

Micrografts containing progenitor cells have been shown to induce a faster evolution of the wound healing process [49]. Animals treated with micrografts present at 3 days a histological appearance similar to those at 7 days that did not receive the micrograft.

The presence of neutrophils in the proliferation and remodeling phases has been shown to be a characteristic of diabetic animals, which was different from the control group. This finding may be due to the greater number of inflammatory cytokines in diabetics, which attract a greater number of neutrophils to the lesion.

The skin wound samples for histopathological analysis were collected three, seven, and fourteen days after creating the artificial wounds for several reasons, which can be justified based on the wound healing process and recent scientific studies. Firstly, this multi-time-point approach allows for a more comprehensive assessment of the dynamic changes that occur during different phases of wound healing. The three-day time stop captures the early inflammatory phase, characterized by acute inflammation, immune cell infiltration, and the initiation of tissue repair processes. By collecting samples at this time point, the initial response to the wound and the early cellular and molecular events can be monitored. The seven-day time stop represents the proliferative phase, during which granulation tissue formation, angiogenesis, and collagen synthesis occur. It is a crucial period for cell proliferation, migration, and extracellular matrix deposition. Collecting samples at this time point enables the evaluation of tissue regeneration and the progression of healing. The fourteen-day mark corresponds to the remodeling phase, characterized by collagen remodeling, wound contraction, and the maturation of the newly formed tissue. By collecting samples at this time point, the structural and functional changes that occur during this critical phase of wound healing can be assessed [25,50,51].

The quantification of collagen production through staining showed that collagen production was more effective in animals treated with micrografts at 3 days of healing compared to those at 7 and 14 days of evolution of control animals. The elastic fibers present in the skin showed no significant difference among the groups studied with regard to fiber production elasticity.

Angiogenesis is the process by which new blood vessels are formed from preexisting vessels in the tissue. VEGF is a potent stimulator of this process, and increased expression of VEGF indicates that blood vessel formation is active, which is important for cell growth and proliferation as well as the formation of granulation tissue. In this study, it was found that at 3 days of healing, VEGF was increased in the groups treated with micrografts compared to the untreated groups, indicating an earlier recovery of vascularization in the CM and DBM groups and favoring faster healing.

Overall, micrografts containing progenitor cells have been shown to accelerate and produce collagen in greater quantities than the control group with longer healing time, resulting in scar tissue with better collagen organization.

Cytokines are proteins that bind to cell membrane receptors and induce their biological effects. The action of cytokines can be autocrine, paracrine, or endocrine. TNF-α is a cytokine produced by macrophages, lymphocytes, and monocytes. Its main physiological effect is to promote immune and inflammatory responses through the recruitment of neutrophils and monocytes to the site of infection and the activation of these cells [52]. TNF-α is present in chronic wounds, such as those present in diabetic feet. In wounds that heal under normal conditions, TNF-α decreases as the cells that produce it are eliminated from the inflammatory process. However, in the wounds of diabetic feet, TNF-α tends to remain at high levels. The treatment with micrografts showed a reduction in TNF-α in diabetic animals compared to those who were also diabetic but did not receive treatment. Thus, the applied micrograft helped to reduce this cytokine, and, consequently, its inflammatory effects.

In addition to pro-inflammatory cytokines, anti-inflammatory cytokines are also important regulators of healing. IL-4 is a cytokine with anti-inflammatory characteristics that is produced mainly by mast cells, eosinophils, Th2 cells, and basophils. IL-4 works in tissue homeostasis by changing macrophages from the M1 profile (pro-inflammatory) to the M2 profile (anti-inflammatory) [53]. Up to 7 days of healing, IL-4 maintained its highest levels in the groups treated with the micrograft. In this phase, inflammatory cells were present from the wound, including macrophages. Therefore, IL-4 may have activated the macrophage alternative pathway for the M2 profile, contributing to the reduction of inflammation. IL-4 is also able to promote the repair of epithelial wounds in vitro by reducing the cytokine-induced epithelial barrier defects.

The importance of IL-10 in wound healing lies in its limitation and termination of inflammatory responses. IL-10 inhibits the infiltration of neutrophils and macrophages into the lesion, as well as the expression of various pro-inflammatory chemokines and cytokines [54]. In this study, at 3 and 7 days after injury, there was no significant difference in IL-10 production among the groups studied. Only at 14 days of healing was IL-10 increased in the groups treated with micrografts compared to the untreated groups. The two together, IL-4 and IL-10, being anti-inflammatory in nature, maintain a favorable environment for healing throughout the healing process. IL-10 is also capable of inhibiting TNF-α. In fact, IL-10 was higher at 14 days in the micrograft-treated groups at the same time that TNF-α was lower in these same groups.

Oxidative stress is the result of an increase in reactive oxygen species (ROS) and/or reactive nitrogen species (RNS) as a result of a constant imbalance between the production of reactive molecules (mainly ROS and RNS) and antioxidant agents [55]. The level of TBARS is pointed out as an effective method to identify these radicals, and high levels of TBARS are present in patients with complications of diabetes mellitus. The level of TBARS was shown to be significantly increased in the DB group compared to the DBM and CM groups.

4. Materials and Methods

4.1. Animal Model

Fifty male Wistar rats (90 days old, weighing around 300 ± 30 g) from the Department of Animal Morphology and Physiology, Rural Federal University of Pernambuco, were involved in this study. The project was submitted to the institutional ethics committee and approved under protocol number 23082.014335/2018–85 and license No. 89/2018.

These animals were kept in cages, with food and water ad libitum, at a temperature of 22 ± 1 °C and with artificial lighting that established a photoperiod of 12 h of light and 12 of dark hours, considering the light period from 06:00 to 18:00 h. The animals were randomly divided into 4 groups, namely: control (C) (n = 10); control treated with micrografts (CM) (n = 10); diabetic (DB) (n = 15); and diabetic treated with micrografts (DBM) (n = 15).

4.2. Diabetes Induction

Diabetes was induced in the animals of the DB and DBM groups by intraperitoneal administration of Streptozotocin solution (Sigma Chemical Co., St. Louis, MO, USA) after a 14 h fasting period. Diabetes was confirmed on the seventh day after the application. Streptozotocin was diluted in 10 mM sodium citrate buffer (pH 4.5) and administered in a single dose of 60 mg/kg of animal weight. The C and CM groups received, in the same way, equivalent doses of saline solution. After 30 min from administration, all animals were fed normally [56].

Only animals with blood glucose above 200 mg/dL (Accu-Chek Activ Kit Glucometer, Roche Diabates Care, Indianapolis, IN, USA) were included in this study, except for the control groups.

4.3. Wound Preparation

The lesions were performed on the backs of the left hind legs of the rats, with a sterile scalpel, in order to remove the skin and subcutaneous tissue until exposure of the muscle tendons (Figure 14).

4.4. Autologous Micrografts Rigenera^® Technology

AMGs were obtained utilizing a Class II medical device, called Rigeneracons, that consists of a grid with six micro-blades encircling hundreds of holes that are 80 µm in diameter, allowing the selection of AMGs with exact sizes using a dimensional exclusion method. An electrical motor, known as Sicurdrill, drives the mechanical fragmentation (Figure 15). Many soft and hard tissues, including the dermis, cartilage, bone, fat, and heart tissues, may be swiftly and effectively broken down using the technology. The AMG solution can be injected around the margins and the bed of a wound or used to imbibe a dermal substitute. Because the AMG technology does not require the use of enzymes or other chemicals, the operation takes only 30 min to complete.

4.5. Histopathological Analysis

The animals in both the experimental and control groups were sedated with ketamine hydrochloride (80 mg/kg) and xylazine (6.0 mg/kg) intramuscularly three, seven, and fourteen days after the artificial wounds creation. The wound skin was collected by a rectangular incision around the lesion (Figure 16). The rats were then euthanized with 100 mg/kg sodium thiopental.

Half of the material was fixed in buffered formalin for 48 h before being paraffin embedded. The sections were stained with hematoxylin and eosin, Mallory’s trichrome, and nitric orcein for histological investigation using a light microscope (OLYMPUS BX-50, Tokyo, Japan). The other half was frozen at −20 °C in a freezer.

Samples for histological analysis were collected at three, seven, and fourteen days following the injury.

4.6. Collagen and Elastic Fibers Quantification

Histological slices were stained with Mallory’s trichrome and nitric orcein, respectively, to quantify the collagen and elastic fibers. For this purpose, three slides were utilized for each group, with five fields captured on each slide. The images were captured with a Sony^® (Tokyo, Japan) video camera attached to an Olympus^® (Tokyo, Japan) Bx50 microscope and submitted to the Gimp 2.0 application for quantification using the RGB Histogram (Red-Green-Blue), which is based on luminescence intensity and where the tones of the image pixels vary from 0 to 255, with tone 0 representing absolute darkness (lowest luminescence) and tone 255 representing absolute white (higher luminescence) [57].

4.7. Immunohistochemical Analysis for COL1, COL3, VEGF-A, IL4, and IL10

COL1A1 (sc-293182, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COL3A1 (sc-271249, Santa Cruz Biotechnology, Santa Cruz, CA, USA), VEGF-A (MBS2540134, MyBioSource, San Diego, CA, USA), IL-4 (sc-53084, Santa Cruz Biotechnology, Santa Cruz, CA, USA), and IL-10 (sc-365858, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies in a 1:100 dilution ratio were used.

The slides were deparaffinized and rehydrated in xylene and alcohols, respectively. The antigen recovery was carried out in the microwave for 5 min at high temperature with a citrate buffer solution (pH 8.0). Endogenous peroxidase was inhibited by a 3% hydrogen peroxide in methanol solution [58]. The nonspecific antigen–antibody interaction was inhibited by incubating the slides in PBS and 5% bovine serum albumin (BSA) for 1 h. All antibodies were diluted in PBS/BSA 1% overnight. The parts were then treated with Histofine^® (Cod. 414191F—Nichirei Biosciences, Tokyo, Japan) for 30 min. The antigen–antibody reaction was detected as a brown precipitate after four minutes of treatment with 3,3 diaminobenzidine and counterstained with hematoxylin. A video camera (Sony) coupled with the Olympus BX-50 microscope was used to collect the images.

4.8. Evaluation of the Oxidative Stress—Measurement of Gluthatione (GSH)

The skin’s oxidative stress was assessed by measuring lipid peroxidation and GSH levels. The quantities of acid reactive chemicals thiobarbiturate (TBARS) were used to estimate lipid peroxidation (Figure 17), whereas non-protein sulfhydryl groups were used to estimate reduced GSH (Figure 18) [59,60]. Skin pieces were macerated in 1.15% KCl in a proportion of 10 mL/1 g until completely homogenized. The homogenate was transferred to a test tube, and 2 mL of the reagent (0.375% thiobarbituric acid and 75% acid trichloroacetic acid) was added for every mL of the mixture. Duplicate tubes were sealed and heated in a water bath (100 °C) for 15 min. The supernatant was separated, and the absorbance was measured at 535 nm [61].

4.9. Evaluation of Tumor Necrosis Factor-α (TNF-α)

TNF-α dose was determined using the ELISA method, according to the manufacturer’s instructions (RAB0479-1KT—Sigma-Aldrich (St. Louis, MO, USA)) [62].

4.10. Statistical Analysis

Statistical analysis was conducted using the Kruskal–Wallis test with post hoc Dunn analysis to assess collagen production and quantification of elastic fibers utilizing Gimp 2.0 software. The significance level was set at p < 0.05.

Regarding collagen I quantification, pixel-based analysis was performed on skin tissue samples from the animal groups. Tukey and Kramer Multiple Comparisons tests showed no significant differences (p > 0.05) among means labeled with the same letter.

Similarly, pixel-based quantification of collagen III, VEGF, IL4, and IL10 in skin tissue samples showed no significant differences (p > 0.05) among means marked with the same letter.

For GSH and TBARS values, statistical significance was determined using the Kruskal–Wallis test with Dunn’s post hoc analysis (p < 0.05). Means sharing the same letter were not significantly different from each other.

The analysis of TNF-α values in the skin of animals across different experimental groups and treatment times was performed using the Kruskal–Wallis test with Dunn’s post hoc test (p < 0.05). Means with the same letter were not significantly different from each other.

The software used for statistical analysis was GraphPad Prism 9.0.0.

5. Conclusions

Autologous micrografts have demonstrated effectiveness in promoting healing of difficult-to-heal wounds by influencing epithelialization, angiogenesis, and collagen production. Treatment with AMG has been observed to reduce tissue inflammation, resulting in improved healing in animals with and without diabetes.