Effect of local transplantation of cultured macrophage loaded in mesenchymal stem cell/ macrophage culture supernatants on wound healing in rat

doi:10.21203/rs.3.rs-4129567/v1

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Effect of local transplantation of cultured macrophage loaded in mesenchymal stem cell/ macrophage culture supernatants on wound healing in rat

https://doi.org/10.21203/rs.3.rs-4129567/v1

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Background: Wound healing is an orderly sequence of events restoring the integrity of the damaged tissue and consists of inflammatory, proliferation, and remodeling phases. The objective of the current study was to investigate the effect of local transplantation of cultured macrophage loaded in mesenchymal stem cell/macrophage culture supernatants on wound healing.

Results: Sixty-four healthy adult male Wistar rats were randomized into 4 groups of sixteen animals each: 1) SHAM group. 2) MAC-MSC/SN group: One milliliter application of a mixture comprising mesenchymal stem cell and macrophage culture supernatants in a 1:1 ratio was administered locally to the wound bed. 3) MAC group: Local transplantation of one million macrophage cells cultured in the wound bed. 4) MAC + MAC-MSC/SN group: Local transplantation of cultured macrophage in combination with mesenchymal stem cell/ macrophage culture supernatants in the wound bed. An incisional wound model was used for biomechanical studies, while an excisional wound model was used for biochemical, histopathological, and planimetric assessments. The wound area was significantly reduced in the MAC + MAC-MSC/SN group compared to other groups (P > 0.05). Biomechanical measurements from the MAC + MAC-MSC/SN group were significantly higher compared to other experimental groups (P > 0.05). Biochemical and quantitative histopathological analyses revealed a significant difference between MAC + MAC-MSC/SN and other groups (P > 0.05). MAC + MAC-MSC/SN showed the potential to improve wound healing significantly. This appears to work by angiogenesis stimulation, fibroblast proliferation, inflammation reduction, and granulation tissue formation during the initial stages of the healing process. This accelerated healing leads to earlier wound area reduction and enhanced tensile strength of the damaged area due to the reorganization of granulation tissue and collagen fibers.

Conclusion: Local transplantation of cultured macrophage loaded in mesenchymal stem cell/macrophage culture supernatants had potential advantages in wound healing.

Cell culture

Macrophage

Mesenchymal stem cell

Supernatant

Wound healing

Skin is the largest organ of the body, and consists of many cell types that work together to maintain homeostasis (1). Although there are differences such as the time for repair to be completed and among tissue types and species, it is important to note that all wounds undergo similar repair processes (2–5). Cutaneous wound healing is a vital physiological process that relies on the coordinated efforts of various cell types and their secreted factors. This intricate interplay is crucial for effective tissue repair (6). The cellular and biochemical events in wound healing can be divided into three distinct phases: the inflammatory response, cellular proliferation and synthesis of extracellular matrix components and the last stage known as remodeling. Significant overlap exists among each phase (7, 8). Effective wound healing relies on essential biologically active polypeptides that regulate wound healing by modifying target cell growth, differentiation, and metabolic activities. These signaling molecules encompass growth factors, cytokines, and chemokines, pivotal in orchestrating the intricate mechanisms underlying tissue repair (9).

Mesenchymal stem cells (MSCs) are extensively studied and known for their remarkable multipotency, enabling them to undergo differentiation into a variety of specialized cell types involved in tissue formation. The cell lineages include osteoblasts, adipocytes, chondrocytes, tenocytes and myocytes (10, 11). Furthermore, beyond their multilineage differentiation potential which plays a crucial role in regeneration, MSCs can modulate immune and inflammatory responses. Also, MSCs possess reparative capabilities through paracrine signaling as they release biologically active molecules that influence surrounding cell migration, proliferation and survival (12). MSCs release a supernatant containing a mixture of growth factors and cytokines, contributing to critical stages and activities in the wound-healing process including the stimulation of angiogenesis, inflammation reduction, facilitation of cell migration and differentiation, and collagen production and remodeling (13, 14). Analysis of the MSC-conditioned medium disclosed the presence of numerous growth factors such as epidermal growth factor (EGF) which holds a crucial role in skin wound reepithelization, fibroblast growth factor (FGF), which promotes cell survival, insulin-like growth factor (IGF), playing a part in wound closure, and vascular endothelial growth factor (VEGF), serving as a primary controller of angiogenesis. Immunomodulatory cytokines like IL-6 and IL-8 have been found in stem cell supernatants and have been shown to contribute to angiogenesis and anti-inflammatory effects (15). MSC-conditioned medium also contains inflammatory regulatory chemokines like macrophage chemoattractant protein-1 (MCP-1), matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) which play a role in tissue remodeling (16). Therefore, administrating an MSC-conditioned medium containing these bioactive molecules rather than whole cells, for wound treatment appears to be a cheaper and safer approach (17).

Macrophages are the main components in all wounds as they contribute to the inflammation necessary to eliminate potential pathogens, resolve inflammation after pathogen clearance and initiate and maintain tissue repair and regeneration (18). Macrophages are the primary inflammatory cells that control the essential molecular signaling pathways for wound repair mechanisms by producing and releasing oxygen-free radicals, inflammatory cytokines and tissue growth factors (19). Around 48 to 72 hours after the injury, the blood monocytes that arrived in the wound with the neutrophils undergo phenotypic changes, influenced by TGF-β, transforming into wound macrophages. These macrophages have a longer lifespan compared to neutrophils. While both cell types have a key role in micro-debridement of the wound, macrophages persist and act as a rich source of cytokines like IL-1, IL-6, IL-8, TNF-α, and potent growth factors such as fibroblast growth factors, epidermal growth factor, TGF-β, and platelet-derived growth factor. Macrophages phagocytize deteriorating neutrophils and also induce neutrophil apoptosis. As a result, within approximately 48 to 96 hours after the injury, macrophages become the predominant leukocyte in the uninfected wound. Macrophages within the wound continue to phagocytose bacteria and debris, releasing matrix metalloproteinases (MMP-1, MMP-2, MMP-3, and MMP-9). These enzymes break down the provisional extracellular matrix and promote cell migration within the wound as a vital aspect of the proliferative phase (2, 5, 20–24). The present study was conducted to evaluate the synergistic effects of local transplantation of cultured macrophage loaded in mesenchymal stem cell/macrophage culture supernatants on wound healing in rat.

2.1 Animals

In this study, 64 healthy adult male Wistar rats, between 8 and 10 weeks and weighing within the range of 200 grams, were employed. All animals were provided compassionate care by a well-trained expert, adhering to the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals, as outlined by the National Institute of Health (NIH publication No. 85 − 23, revised 1985). The animals were individually housed in cages in a controlled experimental room with a natural light/dark cycle. Environmental conditions were set to a constant room temperature of 23 ± 2°C. The rats were fed with standard pellets and had continuous access to water and food.

Surgical procedures were performed under general anesthesia induced through the intraperitoneal injection (IP) of a combination of xylazine hydrochloride 2% (5 mg/ kg, Alfasan International, Woerden, Holland) and ketamine hydrochloride 10% (80 mg/kg, Alfasan International). After the wound creation, the rats were randomly allocated into four groups each consisting of 16 rats. Further subdivisions were made within each group: three subgroups, each comprising four rats for planimetric evaluations; eight were assigned for pathological and biochemical assessments, and four were earmarked for biomechanical evaluations. Four experimental groups (n = 16 rats/group) were randomized as follows: 1) SHAM group. 2) MAC-MSC/SN group: One milliliter application of a mixture comprising mesenchymal stem cell and macrophage culture supernatants in a 1:1 ratio was administered locally to the wound bed. 3) MAC group: Local transplantation of one million macrophage cells cultured in the wound bed. 4) MAC + MAC-MSC/SN group: Local transplantation of cultured macrophage in combination with mesenchymal stem cell/ macrophage culture supernatants in the wound bed. On the final day of the sampling, the rats were euthanized through intraperitoneal administration of a ketamine-xylazine combination. Euthanasia was carried out using a dosage five times more than that of the anesthesia induction dose (25). In this study, all animals, experimental units or data points were included in the analysis. No unexpected adverse effects were observed in rats during the study.

2.2 Macrophage isolation and culture

Rat macrophages were isolated and cultured using a method previously outlined with some modifications (26, 27). After euthanasia, the rats were immersed in 75% alcohol for disinfection. The peritoneal cavity was filled with 15 ml of cold sterile phosphate-buffered saline (PBS). After a gentle 2-minute massage of the abdominal wall, the peritoneal fluid, containing resident macrophages, was collected. The collected fluid was centrifuged at 1,500 rpm for 5 minutes. After discarding the supernatant, the cells were subjected to two sequential washes with RPMI-1640 medium (Sigma Chemical Co., Munich, Germany) enriched with 10% fetal bovine serum (FBS) (10% Gibco. Germany) and resuspended in RPMI-1640 containing 10% FBS. The trypan blue dye exclusion assay assessed the survival rate of peritoneal cells. Subsequently, they were inoculated into a T-75 culture flask and placed in an incubator (ICO105T2K6, Memmert, Germany) set at 37°C with 5% CO2. Following a 2-hour incubation, nonadherent cells were washed out using ice-cold PBS. Adherent macrophages were cultured in RPMI-1640 medium containing 10% bovine serum, penicillin 100U/mL, and streptomycin 100U/mL at 37°C within a humidified environment with 5% CO2. Following 4 days of incubation period, the cells were detached by trypsinization. Cells were counted in Neubauer's chamber using trypan blue exclusion. The viability was never below 95%. Macrophages were qualitatively analyzed through morphological observation with an inverted optical microscope (Fig. 1).

2.3 Mesenchymal stem cell isolation and culture

The extraction of MSCs from bone marrow (BM) was conducted by a previously outlined protocol with some modifications (28). The rats were euthanized and care was taken to remove the skin from their femurs and tibia. The diaphysis of the bone was scraped to remove muscle and connective tissue from the tibia and femur. The bones were immersed in 10% ethyl alcohol for sterilization. The sharp scissors were used to cut the tip of each bone, and the marrow was harvested by introducing a 20-gauge syringe needle into one end of the bone, flushing it with Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Chemical Co., Munich, Germany), and collecting it in a 15-ml tube. It was repeated several times. The bone marrow cells were filtered through a 70-mm nylon mesh filter before proceeding to the next step. All these processes were performed in a chilled environment on ice. BM cells were cultured in DMEM supplemented with 10% FBS (10% Gibco. Germany) within a 25 cm² culture flask, followed by incubation (ICO105T2K6, Memmert, Germany) at 37°C with 5% CO2. Following 24 h incubation, the supernatants of cell culture were then replaced with fresh medium without serum and incubated for 48 hours. The conditioned media were filtered through a 0.22 µm filter, and stored at -80°C until use. (Fig. 1-B) (Fig. 2)

2.4 Excisional Wound Creation

After anesthesia induction, the rat's back hair was clipped and the surgical site was prepared by scrubbing. Excision wounds were created in the rats by removing a 20mm diameter circular, full-thickness section of skin from the dorsal thoracic region, utilizing a size 10 surgical blade. To achieve this, we employed the protocol outlined by Ferreira et al. in 2008, positioning the rats in a lateral recumbency (29).

2.5 Incisional Wound Creation

After anesthesia induction and surgical site preparation, a 4-cm full-thickness skin incision was made along the midline of the dorsal surface. Following the local administering of the treatment to each experimental group, the incision site was closed using 4 − 0 nylon sutures at half-centimeter intervals, employing a simple interrupted pattern.

2.6 Treatment

According to previous research, a predetermined quantity of specific biological materials tailored for each experimental group was uniformly injected at consistent coordinates across all wounds (30, 31). In the macrophage treatment group, cells were suspended in 1 ml of PBS before administration. The MAC + MAC-MSC/SN group received an injection of cells suspended in 1 ml of a 1:1 (v/v) mixture of mesenchymal stem cell and macrophage culture supernatants. For local administration in the excisional wound, a 1 ml cell suspension and, or supernatants were subcutaneously injected at four sites around the wound bed using a syringe, maintaining a distance of 3 mm from the rim. In the case of the incisional wound, cell suspension and, or supernatants was applied the same way in the excisional wound model. More precisely, the formulations were injected at four specific points along the incision, maintaining uniform intervals between each application site. The injection was administered at a single dose immediately after wound creation (day 0).

2.7 Wound Area

Assessing wound healing efficacy involved determining the percentage reduction in the wound area and the time for wound closure. To assess the excisional wound contraction and closure rate, photographs were taken immediately after the wound was created and on days 3, 6, 9, 12, 15, 18, and 21 post-operations using a digital camera while a ruler was positioned adjacent to the wounds to provide a scale reference. The Measuring Tool of Adobe Acrobat 9 Pro Extended software (Adobe Systems Inc, San Jose, CA) was used to measure the wound areas (32).

2.8 Biomechanical Evaluations

On the ninth day after creating an incisional wound, the sutures were removed, and a strip of skin was taken from the rat using Metzenbaum scissors, ensuring that the wound was positioned in the middle of the strip. The sample was subsequently enveloped in gauze soaked in Ringer's solution, sealed within plastic bags, and stored in a freezer at -20°C until mechanical testing. The Santam STM-5 Universal Testing Machine was used for the assessment (Santam Co., Tehran, Iran). The skin strips were equipped with suitable clamps, and the initial distance between the clamps at the beginning of the test depended on the sample size. Each sample was stretched at a constant speed of 20 mm/min and this stretching continued until complete tensile failure.

2.9 Biochemical Evaluations

To conduct biochemical assessments, samples were collected from the tissue concurrently with pathological samples on day 21. These samples were promptly frozen at -20 degrees. The tissues were ground in a mortar. One gram of each tissue sample was homogenized on ice with 4.5 ml of the appropriate buffer using an Ultra-Turrax homogenizer (IKA, Werke, Germany) for 15 minutes. Filtered homogenates were subjected to centrifugation using a refrigerated centrifuge at 4°C. Subsequently, the resulting supernatants were utilized for assessing enzymatic activities. All assays were conducted at room temperature. The supernatant's Total Antioxidant Capacity (TAC), Total Oxidant Status (TOS), and Malondialdehyde (MDA) levels were measured by a spectrophotometer using commercial assay kits (Navand Salamat, Tehran, Iran), adhering to the manufacturer's guidelines. Following a previously established protocol, the supernatant derived from samples collected on day 21 was utilized to assess glutathione peroxidase (GPx) activity (33). The procedure involved the introduction of KH2PO4, EDTA, GSH, B-NADPH, NaN3, and GR into the sample, followed by an incubation period. Upon the addition of H2O2, the chronometer was initiated, and the absorbance at 340 nm was monitored at 15-second intervals for 5 minutes. On postoperative day 21, a skin sample was gathered from the fully healed wound area and examined for hydroxyproline content, a fundamental component of collagen. Estimating hydroxyproline levels followed a method outlined by previous researchers (34). In summary, the tissues were dried in a hot-air oven set at 60°C to 70°C until a consistent weight was achieved. Subsequently, they underwent hydrolysis in sealed tubes with 6 N HCl at 130°C for 4 hours. The resulting hydrolysate was neutralized to pH 7.0 and underwent a 20-minute chloramine-T oxidation process. The reaction concluded with adding 0.4 M perchloric acid, and the color development occurred through applying Ehrlich reagent at 60°C. The measurement was conducted at 557 nm using a UV-visible spectrophotometer (CamSpec M330).

2.10 Histopathological Evaluations

Skin samples were obtained from the wound periphery, along with normal skin, at 7, 14, and 21 days post-excisional wound creation. These samples were then fixed in 10% formalin and embedded in paraffin for subsequent histopathological investigation using a light microscope. Hematoxylin and eosin-stained slides were scrutinized to assess cellular infiltration, encompassing the count of mononuclear cells, polymorphonuclear cells, and fibroblastic aggregation. Additionally, the examination involved quantifying the distribution of blood vessels within one square millimeter of the wound, along with evaluating the thickness of the epithelial tissue in the repair region.

2.11 Statistical Analyses

The Kruskal-Wallis variance analysis was adopted for differences among groups. Where the P value (from the Kruskal-Wallis test statistics) was statistically significant, multiple comparison tests were utilized to get the differences. Mann-Whitney U test was used for comparison among days. For retrieving possible multiple comparisons, the Bonferroni correction was utilized. We utilized SPSS 11.5 (SPSS Inc) for the analyses and considered a P value < 0.05 as a significant level.

3.1 Wound area reduction

The effect of macrophage, mesenchymal stem cell/macrophage culture supernatants, and macrophage + mesenchymal stem cell/macrophage culture supernatants on circular excision wound contraction area is shown in Table 1. Local transplantation of MAC + MAC-MSC/SN significantly reduced the wound area and enhanced the healing rate compared to other groups ( P < 0.05) (Fig. 3).

Table 1

Effect of macrophage, mesenchymal stem cell/macrophage culture supernatants on circular excision wound contraction area (mm²). Values are given as mean ± SEM.
Groups	Day 3	Day 6	Day 9	Day 12	Day 15	Day 18	Day 21
SAHM	314.77 ± 9.17	252.25 ± 6.75	103.55 ± 6.80	68.11 ± 3.65	49.15 ± 2.55	20.25 ± 1.23	9.88 ± 0.25
MAC-MSC/SN	237.98 ± 8.65*	159.76 ± 5.65*	67.35 ± 5.87*	52.62 ± 3.27*	27.65 ± 2.76*	13.29 ± 1.11*	5.70 ± 0.66*
MAC	229.260 ± 9.80*	154.16 ± 5.81*	62.12 ± 4.54*	49.64 ± 3.23*	31.18 ± 2.22*	14.695 ± 1.12*	4.13 ± 0.50*
MAC + MAC-MSC/SN	98.13 ± 7.25**	75.74 ± 4.71**	45.98 ± 4.15**	19.47 ± 0.25**	6.237 ± 1.09**	3.11 ± 1.03**	1.67 ± 0.05**
The mesenchymal stem cell and macrophage culture supernatant treated group was compared by Student t test with other groups. ,*: The mean difference is significant at the .05 level vs other groups.

3.2 Biomechanical Findings

Biomechanical parameters including stress, strain, maximum stored energy (MSE), stiffness, and ultimate strength measured from the macrophage + mesenchymal stem cell/ macrophage supernatants group were significantly higher compared to other experimental groups ( P < 0.05) (Table 2). These assessments indicated that administration of MAC + MAC-MSC/SN showed significantly better biomechanical properties in treated incisional wounds ( P < 0.05).

Table 2

Biomechanical parameters assessed for each of the experimental groups. Values are given as mean ± SEM.
Groups	Stress (Mpa)	Strain (%)	Ultimate Strength (N)	Stiffness ( Mpa)	MES (J)
SAHM	0.14 ± 0.30	41.80 ± 3.57	12.30 ± 3.57	0.36 ± 0.75	8.14 ± 1.17
MAC-MSC/SN	0.25 ± 0.50	43.78 ± 3.91	17.36 ± 2.25	0.32 ± 0.98	12.35 ± 1.15
MAC	0.24 ± 0.43	40.35 ± 3.24	17.11 ± 1.13	0.29 ± 0.96	11.70 ± 1.22
MAC + MAC-MSC/SN	0.26 ± 0.67*	52.93 ± 3.77*	19.98 ± 1.19*	0.52 ± 0.76*	21.48 ± 4.32*
MES: Maximum Energy Store. *: The mean difference is significant at the .05 level vs SHAM, MAC-MSC/SN and MAC

3.3 Hydroxyproline contents

Contents of hydroxyproline in the SHAM, MAC-MSC/SN, MAC, and MAC + MAC-MSC/SN groups were recorded as 52.71 ± 5.89, 65.36 ± 4.92, 67.39 ± 4.81 and 89.73 ± 4.34 mg g-1, respectively. The concentrations were significantly bigger in the MAC + MAC-MSC/SN group implying further deposition of collagen in comparison to other groups (P < 0.05).

3.4 Biochemical evaluation of healed wound area

The levels of antioxidant enzymes (GPx), the natural product of lipid peroxidation (MDA), total antioxidant capacity (TAC), and total oxidant status (TOS) were estimated in this study to evaluate the effect of cultured macrophage in combination with mesenchymal stem cell/ macrophage culture supernatants on wound-healing progress (Table 3). Biochemical analyses revealed that TAC and GPx activity values in the MAC + MAC-MSC/SN group were significantly higher than those observed in the other experimental groups. In contrast, MDA and TOS levels significantly decreased in the MAC + MAC-MSC/SN group compared to the other groups (P < 0.05).

Table 3

Comparison of the activities of TAC, TOS, MDA, and GPx in the tissue samples taken from experimental groups on day 21. Data are expressed as Mean ± SD.
Indices	SAHM	MAC-MSC/SN	MAC	MAC + MAC-MSC/SN
TAC	0.56 ± 0.05	0.81 ± 0.17	0.85 ± 0.15	1.17 ± 0.87*
TOS	102.90 ± 18.50	59.79 ± 8.21	55.19 ± 7.35	37.61 ± 12.59*
MDA	0. 95 ± 0.33	0.57 ± 0.18	0.53 ± 0.14	0.35 ± 0.17*
GPx	0.11 ± 0.08	0.28 ± 0.03	0.25 ± 0.05	0.51 ± 0.13*
TAC: Total antioxidant capacity, TOS: Total oxidant status, MDA: Malondialdehyde, GPx: Glutathione peroxidase dismutase. * P < 0.05 vs. MAC-MSC/SN and MAC.

3.5 Histopathological evaluation of healed wound area

The histopathology of the wound tissues was evaluated on the 7th, 14th, and 21st day after wound creation (Fig. 4). The number of PMN cells and mononuclear cells in the MAC + MAC-MSC/SN group was significantly less compared to those in the other experimental groups at all time points ( P < 0.05) (Fig. 5,6). The number of blood vessels and fibroblast cells was significantly higher in the MAC + MAC-MSC/SN group on days 7 and 14 compared to the other groups ( P < 0.05). Conversely, the number of blood vessels and fibroblasts measured from the samples taken on day 21 was significantly decreased in this group compared to the other experimental groups ( P < 0.05) (Fig. 7,8). The thickness of the granulation tissue on day 7 was significantly higher in the MAC + MAC-MSC/SN group than the other groups, while on days 14 and 21, the granulation tissue was thicker in the MAC-MSC/SN and MAC groups than in the MAC + MAC-MSC/SN group (Fig. 9) (P < 0.05).

The advancement in wound healing technology has enabled the use of cells to address the limitations of conventional methods. Cell therapy can enhance wound healing without major surgery and donor site complications. In acute wound management, cell therapy can accelerate wound healing, reduce scar contraction, and minimize donor site complications. Furthermore, this technique is simple and time-saving, reducing patients' surgical burden. Currently, in clinical practice, keratinocytes, fibroblasts, bone marrow stromal stem cells and adipose-derived stromal vascular fraction (SVF) cells are used (35). It is known that allogeneic cells do not permanently attach to the wound and are quickly replaced by host cells. However, allogeneic cells stimulate migration and proliferation of host cells from the wound beds and edges because they release growth factors, extracellular matrices and basement membrane components. Eventually, allogeneic cells play an effective role in accelerating epithelialization and production of granulation tissue (36). Because of the rich presence of cytokines and growth factors, the mesenchymal stem cell culture supernatant exhibits beneficial effects on the healing process (13). It was also revealed that wound healing can be accelerated by increasing the number of macrophages or monocytes at the wound site through the delivery of pullulan-collagen hydrogel scaffolds (37).

Assessing wound contraction is an indicator of understanding the progress of skin wound healing. Wound contraction accelerates the healing rate by limiting the amount of granulation tissue that needs to be produced (38). Wound contraction begins shortly after wound formation and reaches its maximum within 2 weeks with a considerable decrease in wound area (39). Our study revealed that local MAC + MAC-MSC/SN transplantation results in higher wound contraction and accelerated wound closure compared to other experimental groups.

During the inflammatory phase, a large number of radicals are produced due to tissue damage. Radicals are commonly associated with oxidative stress, resulting in lipid peroxidation and compromised wound healing (40). Excessive ROS contribute to the reduction of fibroblasts, keratinocytes, and endothelial cells in the wound healing process. Antioxidants limit ROS levels to prevent oxidative stress-induced cell damage (41, 42). Our study revealed that local transplantation of MAC + MAC-MSC/SN significantly improved the biochemical indices, including a decrease in MDA and TOS and increasing the level of GPx and TAC compared to the other experimental groups. Decreased levels of MDA indicated lipid peroxidation prevention by effectively scavenging free radicals in wounds. The better antioxidant effects at the wound site MAC + MAC-MSC/SN treated group in this study also contributed to improved granulation and healed tissue quality in this group as supported by histopathological findings. Hydroxyproline, a main component of collagen, enables the sharp twisting of the collagen helix. It provides firmness to the collagen's triple-helical structure by forming hydrogen bonds. Hydroxyproline has been used as a reliable indicator for evaluating collagen content because it is present in a few proteins apart from collagen (43). The higher concentration of hydroxyproline in the MAC + MAC-MSC/SN group, as a direct measure of collagen synthesis, demonstrated an increase in collagen deposition. Collagen provides the tensile strength of wounds. Therefore, the MAC + MAC-MSC/SN group in the incisional skin wound model exhibited statistically significant improvements in biomechanical parameters compared to other groups.

Angiogenesis, the development of new blood vessels, is one of the necessities of the wound healing process, which can increase blood supply and subsequently lead to faster healing (44). In the current study, the highest amount of angiogenesis was observed in the MAC + MAC-MSC/SN treatment group. Angiogenesis provides a framework for the creation of connective tissue in the early days of healing. The formed microvessel enables the transfer of fluid, oxygen, nutrients, and immune-competent cells to the wound area (45). Increased formation of new vessels following wound creation indicated that MAC + MAC-MSC/SN could promote the healing process due to facilitating cellular infiltration. On day 21 after injury, the number of blood vessels in the MAC + MAC-MSC/SN group was lower than in other groups. This acceleration in blood vessel reduction indicates the positive effect of MAC + MAC-MSC/SN healing.

The higher production of fibroblasts in the MAC + MAC-MSC/SN group on days 7 and 14 showed that this treatment could induce the growth of fibroblasts. Fibroblast number is a widely recognized index in the assessment of connective tissue healing quality. The primary role of fibroblasts is collagen production (46). Fibroblasts synthesize essential components of the primary extracellular matrix in the wound bed, providing an optimal condition for cell migration and proliferation. Subsequently, fibroblasts produce collagen, which is crucial for imparting tensile strength to the wound bed (47). Fibroblasts measured on day 21 in the MAC + MAC-MSC/SN group had a significantly lower compared to other experimental groups, which could be the result of fibroblasts developing into fibrocytes. Fibroblasts are removed through newly formed blood vessels at the wound site and are transformed into fibrocytes over time. Fibrocytes are developed fibroblasts that can make collagen more than fibroblasts (46). Collagen contributes to the strength and integrity of the tissue matrix and plays a key role in homeostasis and epithelialization during the later stages of healing (48).

Granulation tissue acts as the base, creating a matrix for proper healing. formation of granulation tissue in the early days of the healing process is regarded as a crucial factor in wound healing acceleration (49). Superior granulation tissue is characterized by the presence of well-developed blood vessels in perpendicular directions, along with a dominance of fibroblasts and a well-organized extracellular matrix formation.. In the initial stage, immature-type granulation tissue consists of inflammatory cells, angioblasts, new blood vessels, fibroblasts, and collagen fibers. In the later stages, this immature granulation tissue matures, becoming more permanent, which is essential for effective wound healing. In the current study, the maximum thickness of the granulation tissue was observed in all groups at 7 days after wounding. Moreover, this thickness was significantly greater in the MAC + MAC-MSC/SN group than in the other groups. On days 14 and 21 post-injury, the thickness of the granulation tissue decreased due to maturation. the thickness of the granulation tissue in the MAC + MAC-MSC/SN group was significantly lower compared to other treatment groups.

The initial stage of wound healing begins with an inflammatory phase, characterized by inflammatory cells. This phase is critical in wound healing because of its association with cellular events, contraction, and wound closure (50). The lowest PMN and mononuclear cell count on all time points was observed in the MAC + MAC-MSC/SN group in our study. This observation might be attributable to the antibacterial and anti-inflammatory properties of macrophage loaded in mesenchymal stem cell/macrophage culture supernatants that could lead to a reduction in inflammatory cells through a decrease in secondary infection and accelerate the commencement and cessation of the inflammation (51). Our findings suggested that this treatment improved the wound-healing process by reducing the duration of the inflammatory phase and rapid transition to the proliferative phase, during which increased wound contraction was observed.

In conclusion, our study suggested that MAC + MAC-MSC/SN had potential advantages in wound healing via its antioxidant properties, induction of angiogenesis, proliferation of fibroblast, early formation of granulation tissue, reduction in the number of inflammatory cells, improving collagen deposition, and accelerating wound closure.

MSC Mesenchymal stem cell

EGF Epidermal growth factor

FGF Fibroblast growth factor

IGF Insulin-like growth factor

VEGF Vascular endothelial growth factor

IL Interleukin

MCP-1 Macrophage chemoattractant protein-1

MMP Matrix metalloproteinase

MAC Macrophage

SN Supernatant

Ethics approval and consent to participate

The procedures of this work were approved by the University Ethical Committee and filed under IR-UU-AEC-3/47 code. We followed instructions of National Academy of Sciences Publication with the number 85-23 which was revised in 1985.

Consent for publication

Not applicable

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

The authors declare that they have no competing interests

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article

Authors' contributions

RM and ND designed the studies. NM and RM wrote the manuscript and performed the biomechanical studies. NM performed surgical procedures and sample collection. NM and ND were in charge of cell isolation and culture. R.H. performed the histopathology studies. VM performed the biochemical studies. RM Analyzed the data.

Acknowledgments

We appreciate the Department of Immunology, Urmia University for the technical expertise. The present study was part of a research project performed as a dissertation of the first author (N.M.) submitted as a partial fulfillment of the Degree of DVSc at Urmia University, Urmia, Iran.

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No competing interests reported.

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Effect of local transplantation of cultured macrophage loaded in mesenchymal stem cell/ macrophage culture supernatants on wound healing in rat

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Abstract

Figures

1 Background

2 Methods

2.1 Animals

2.2 Macrophage isolation and culture

2.3 Mesenchymal stem cell isolation and culture

2.4 Excisional Wound Creation

2.5 Incisional Wound Creation

2.6 Treatment

2.7 Wound Area

2.8 Biomechanical Evaluations

2.9 Biochemical Evaluations

2.10 Histopathological Evaluations

2.11 Statistical Analyses

3 Results

3.1 Wound area reduction

3.2 Biomechanical Findings

3.3 Hydroxyproline contents

3.4 Biochemical evaluation of healed wound area

3.5 Histopathological evaluation of healed wound area

4 Discussion

5 Conclusion

Abbreviations

Declarations

References

Additional Declarations

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