Compare the effectiveness of extracorporeal shockwave and hyperbaric oxygen therapy on enhancing wound healing in a streptozotocin-induced diabetic rodent model

Abstract

Studies have revealed that both extracorporeal shock-wave therapy (ESWT) and hyperbaric oxygen therapy (HBOT) can accelerate wound healing. This study aimed to compare the effectiveness of ESWT and HBOT in enhancing diabetic wound healing. A dorsal skin defect in a streptozotocin-induced diabetes rodent model was used. Postoperative wound healing was assessed once every 3 days. Histologic examination was performed with hematoxylin and eosin staining. Proliferation marker protein Ki-67 (Ki-67), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and 8-hydroxy-2-deoxyguanosine (8-OHdG) were evaluated with immunohistochemical (IHC) staining. The wound area was significantly reduced in the ESWT and HBOT groups compared to that in the diabetic controls. However, the wound healing time was significantly increased in the HBOT group compared to the ESWT group. Histological findings showed a statistical increase in neovascularization and suppression of the inflammatory response by both HBOT and ESWT compared to the controls. IHC staining revealed a significant increase in Ki-67, VEGF, and eNOS but suppressed 8-OHdG expression in the ESWT group compared to the HBOT group. ESWT facilitated diabetic wound healing more effectively than HBOT by suppressing the inflammatory response and enhancing cellular proliferation and neovascularization and tissue regeneration.

INTRODUCTION

Chronic wounds occur commonly and reduce the quality of life of those affected, posing a relevant clinical and socioeconomic burden. Diabetic foot ulcers (DFU) are a main complication of diabetes and may be a significant part of diabetic foot. Overall, approximately 8% of diabetic Medicare beneficiaries have a foot ulcer, and 1.8% have received an amputation.^1,2 Appropriate wound management varies according to the cause of the wound and the principles of effective wound care, such as debridement, adequate pressure offloading, treatment of infection, bypass surgery for ischemic ulcers, and topical wound dressings.^3–5 However, some outcome of DFU are still unfavorable. Therefore, the treatment of DFU continues to be a challenge, as there remains ongoing uncertainty concerning optimal approaches to its management.

Hyperbaric oxygen therapy (HBOT) is the use of 100% oxygen in a pressurized environment. In an HBOT chamber, the air pressure is increased two to three times higher than normal air pressure. When this extra oxygen is throughout the body, it might stimulate the release of growth factors and recruit stem cells, which promote wound healing. HBOT meaningfully affects endothelial nitric oxide synthase (eNOS) and reactive oxygen species.⁶ HBOT potentiates diabetic wound healing by promoting fibroblast cell proliferation and collagen synthesis.^7,8 HBOT has been described as an adjuvant therapy to enhance wound healing of DFU for decades.^9–11 Studies reported that HBOT at pressures of 2.0–2.5 atmospheres absolute (ATA) for 20 times could significantly improve the healing of foot ulcers and reduce amputation rates compared with standard therapy.^12,13 These indicate the effectiveness of hyperbaric oxygen (HBO) for chronic DFU.^14,15

Extracorporeal shock-wave therapy (ESWT) is used to treat different pathologies of the musculoskeletal system, including bone nonunion, elbow epicondylitis, plantar fasciitis, and calcified or noncalcified tendonitis.^16,17 Studies have shown that ESWT can effectively stimulate multiple endogenous growth factors, induce angiogenesis, and heal injuries, fractures, and wounds.^18,19 Our past studies showed that ESWT essentially upgraded diabetic wound healing essential to elevated neoangiogenesis and tissue regeneration, as well as local anti-inflammatory responses, in rodent models of streptozotocin (STZ)-induced diabetes and clinical trials.^20,21 Our previous study has demonstrated that two sessions of ESWTs are better than one session of ESWT in enhancing diabetic wound healing in a rodent STZ-induced diabetes model.²² This indicated two sessions of ESWT is the optimal dosage to accelerate wound healing. In contrast, our pilot clinical trials also showed patients in the ESWT-treated group have better results than the HBOT group to increase diabetic wound healing. However, the biomechanisms are still unclear. Therefore, this study aims to compare the effectiveness of HBOT and ESWT using dorsal skin wounding models in rodents and elucidates the biomechanisms of angiogenesis and anti-inflammatory response among these groups.

MATERIALS AND METHODS General

Experiments involving animals abided by the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health, with approval from the Institutional Animal Care and Use Committee on the protection of animals used for scientific purposes (IACUCA animal use protocol approval number: CGMH-2007111902 and KMUH-105090). Animal housing was performed under a 12–12 h light/dark cycle with freely available rodent chow and water.

Animal model

Male Wistar rats (4-month-old) provided by National Experimental Animals Production Center (Taiwan) underwent diabetes induction by one dose intraperitoneal injection of STZ (Sigma-Aldrich, St. Louis, MO.) dissolved in freshly made sodium citrate buffer (0.1 M, pH 4.5) to a final concentration of 65 mg/kg as type I diabetes mellitus model.²⁰ Totally 7 days following the injection of STZ, the rat blood glucose levels from acquired blood samples were tested using a glucometer. The animals showing fasting blood glucose amounts above 300 mg/dL were considered as successful induction of diabetes, and then, these rats were used for subsequent experiments. To glucose level, we subcutaneously administered intermittent-acting insulin (1–2 U/kg; Montards Novo Nordisk A/S, Frankfurt, Germany) to the diabetic rats' regulatory until the animals were killed with an overdose of sodium pentobarbital.

The animal model followed that of our previous reports.^20,22,23 The dorsum of the rat was shaved, and the palpable hip joints were used as anatomical landmarks for defining the base of the wound defect, which consisted of a full-thickness skin defect measuring 6 × 5 cm². The edge of the wound defect was stitched and set up with 4-0 silk stitches to forestall wound contracture.²⁰ The injury was then briefly covered with clear Tegaderm (3M HealthCare, Borken, Germany) until shockwave or hyperbaric oxygen was started. After the operation, the rodents were gotten back to their enclosures in the animal holding room after they had recovered awareness. The rats were housed separately postoperatively. Antibiotics (ampicillin 50 mg/kg/day; Y F Chemical Corp., New Taipei, Taiwan) were administered routinely to these animals for 3 days postoperatively.

Experimental establishment

Male Wistar rodents were randomized into five groups (n = 10 each). Anesthesia was achieved by inhalation of general anesthesia with isoflurane and intramuscular administration of 0.1 mg/kg atropine to lessen salivary discharge during and after the operation. Group I normal control (NC), included nondiabetic rats receiving no treatment. In Group II diabetic control rats (C), including the diabetic rats with the dorsal skin defect were created but received no treatment (Figure 1). In Group III, ESWT, including the diabetic rats with the dorsal skin defect were developed, and then treated with defocused shock waves (MTS Reflector Type CP155, Konstanz, Germany) of 800 impulses at 10 kV, equivalent to an energy flux density of 0.09 mJ/mm² in two sessions on post-wounding Days 3 and 7.²² ESWT was applied to eight regions along the dorsal wound edge (100 pulses/area of all wound edges × 8 regions). The time required to perform 800 extracorporeal shockwave pulses is approximately 20 min. Ultrasound transmission gel (Pharmaceutical Innovations Inc., Newark, NJ) was used as the contact medium between the ESWT device and the skin. Diabetic rodents in group IV (HBOT) received HBO at a pressure of 2.5 ATA (kg/cm²) for 60 min, then day to day for 10 days including the day of biopsy following previous studies,^24–26 with small animals in hyperbaric chambers (Genmall Biotechnology Co., Ltd., Taipei, Taiwan). The rats did not need anesthesia and can breathe spontaneously during HBOT. After HBOT, the animals were placed back into cages.²⁶ Each exposure was begun at the same hour in the morning (10 a.m.) to rule out any confounding issues related to changes in biorhythms.²⁷

Estimation of the wound healing area

The area of wound healing was assessed weekly after surgery using the template technique, which has been described previously.²⁰ By visual inspection, a clear demarcation line of the unhealed tissue area was delineated on transparent graph paper. Cut and measure the tracking area. The area of wound healing, given by the expression 1 − A1/A0 × 100%, is calculated weekly from the original wound area (6 × 5 = 30 cm² as A0) and the unhealed area (A1) until the wound is fully healed.

Immunohistochemical staining and histomorphometric examination

Full-thickness 3 mm biopsies were acquired from wound margins to study pathological changes in the wound on Day 10 or Day 17 after wounding. Biopsy specimens were fixed in 10% formalin (Sigma-Aldrich) and implanted in paraffin. Sections (4 mm thick) from each group were stained with hematoxylin and eosin (H & E; Sigma-Aldrich). Semiquantitative immunohistochemical (IHC) staining was performed utilizing a horseradish peroxidase–diaminobenzidine (HRP–DAB) staining kit (R&D, Inc., Minneapolis, MN) as previously described.²⁰ Polyclonal antibodies against CD45, 8-hydroxy-2-deoxyguanosine (8-OHdG), proliferation marker protein Ki-67 (Ki-67), vascular endothelial growth factor (VEGF), and eNOS (Santa Cruz, Santa Cruz, CA) were utilized as the primary antibodies, and the sections were incubated with these diluted antibodies (1:100) in phosphate-buffered saline for 60 min. The sections were then incubated with biotinylated goat anti-rabbit antibodies for half-hour. HRP is used to visualize the specific binding of the secondary antibody to the primary antibody to enzymatically convert the chromogenic substrate 3,3′ DAB to a brown precipitate. Sections were mounted, cleared, coverslipped, and examined using a Zeiss fluorescence microscope (Zeiss, Gottingen, Germany).

Tissue sections were imaged utilizing a Zeiss Axioskop 2 plus microscope (Carl Zeiss, Gottingen, Germany) to measure immunohistochemically stained cells. All pictures of every example were caught utilizing a Cool CCD camera (SNAP-Pro cf. Digital kit; Media Cybernetics, Silver Spring, MD). Pictures were examined utilizing Image-Pro Plus image analysis software (Media Cybernetics) as described previously.²⁰ Four random images were acquired from each chosen region at 400× magnification. The percentage of positively labeled cells to total cells and the number of immunopositive cells are given. Two researchers, unaware of the treatment regimen, measured all slices at 400× magnification.

Data management and statistical analysis

Data are mean ± standard deviation of the mean from ≥3 replicates for each experiment. One-way analysis of variance and two-tailed student's t-test (two groups) was performed for comparisons. Post hoc comparisons were determined utilizing Tukey's posttest (multiple groups), and p < 0.05 was considered statistically significant.

RESULTS ESWT are superior to HBOT in enhancing diabetic wound healing

The experimental outcomes uncovered that the wound size was significantly decreased during the process of wound healing in the ESWT- and HBOT-treated groups compared to the diabetic control group. The time to total wound healing was essentially quicker in ESWT and HBOT groups than in the untreated diabetic control group (p < 0.001) (Figure 2). Regardless, the wound healing time was significantly longer in the HBOT-treated group than in the ESWT-treated group (7.50 ± 1.05 weeks vs. 6.00 ± 1.67 weeks, p = 0.046). These results indicated that ESWT and HBOT both facilitate wound healing, especially in ESWT could promote faster diabetic wound healing than HBOT.

ESWT and HBOT both suppressed the inflammatory response

Specimens collected from the periwound region were examined histologically. H&E staining showed that the ESWT-treated and HBOT-treated diabetic rat groups had significantly reduced leukocyte infiltration from the dermis to the subcutaneous myometrium on Day 10 after wounding compared with the diabetic control group (Figure 3A). However, the inflammatory response showed no obvious differences on Day 17 after wounding among the ESWT- and HBOT-treated groups and the control group without treatments. The IHC staining indicated that CD45⁺ expression was significantly decreased in the ESWT- and HBOT-treated diabetic rats' groups when compared with rats in the diabetes mellitus (DM) control group (Figure 3B). These results indicated that diabetic rats that received the ESWT and HBOT treatments showed suppression of early inflammatory responses and enhanced diabetic wound healing.

ESWT and HBOT both suppressed 8-OHdG expression

Oxidative damage indicated by 8-OHdG levels indicated that the ESWT and HBOT groups had significantly reduced diabetic wound healing process at Day 10 (36.72 ± 5.23 [ESWT] vs. 83.57 ± 5.72 [diabetic control], p < 0.001; and 46.78 ± 12.33 [HBOT] vs. 83.57 ± 5.72 weeks [diabetic control], p < 0.001) and Day 17 after wounding (23.21 ± 2.49 [ESWT] vs. 63.24 ± 3.35 [diabetic control], p < 0.001; and 40.47 ± 11.32 [HBOT] vs. 63.24 ± 3.35 weeks [diabetic control], p < 0.001) compared to the diabetic control group (Table 1; Figure 4A). In addition, 8-OHdG expression was significantly decreased in ESWT group compared with HBOT group.

TABLE 1 Summary of immunohistochemical staining of CD45, 8-OHdG, Ki-67, VEGF, and eNOS expression at Days 10 (D10) and Days 17 (D17) in normal, control (Ctrl), extracorporeal shock-wave therapy (ESWT), and hyperbaric oxygen therapy (HBOT) treatment.

		Normal	DM
			Ctrl	ESWT	HBOT
CD45	D10	1.31 ± 0.93	9.18 ± 2.12	1.23 ± 0.46	0.38 ± 0.27
	p value	<0.001	Ref.	<0.001	<0.001
	D17	3.43 ± 0.95	3.45 ± 0.44	0.87 ± 0.22	0.62 ± 0.30
	p value	0.48	Ref.	<0.001	<0.001
8-OHdG	D10	52.14 ± 5.21	83.57 ± 5.72	36.72 ± 5.23	46.78 ± 12.33
	p value	<0.001	Ref.	<0.001	<0.001
	D17	42.72 ± 2.52	63.24 ± 3.35	23.21 ± 2.49	40.47 ± 11.32
	p value	<0.001	Ref.	<0.001	0.001
Ki-67	D10	40.42 ± 3.12	16.66 ± 1.99	81.08 ± 3.79	51.41 ± 6.02
	p value	<0.001	Ref.	<0.001	<0.001
	D17	51.30 ± 5.16	19.07 ± 4.22	84.15 ± 3.72	62.74 ± 5.34
	p value	<0.001	Ref.	<0.001	<0.001
VEGF	D10	47.82 ± 4.83	19.15 ± 4.42	66.22 ± 3.67	48.19 ± 3.65
	p value	<0.001	Ref.	<0.001	<0.001
	D17	54.20 ± 3.04	25.39 ± 2.52	85.65 ± 4.15	57.10 ± 7.43
	p value	<0.001	Ref.	<0.001	<0.001
eNOS	D10	48.98 ± 6.17	38.67 ± 2.77	65.14 ± 4.00	54.03 ± 5.21
	p value	0.004	Ref.	<0.001	<0.001
	D17	87.28 ± 3.41	50.60 ± 3.89	93.41 ± 2.78	74.88 ± 5.17
	p value	<0.001	Ref.	<0.001	<0.001

Note: Experimental results are presented as the means ± standard error (SE) and obtained from six specimens.

Abbreviations: 8-OHdG, 8-hydroxy-2-deoxyguanosine; eNOS, endothelial nitric oxide synthase; Ki-67, proliferation marker protein Ki-67; VEGF, vascular endothelial growth factor.

ESWT and HBOT both enhanced cellular proliferation and regeneration

Cell proliferation was assessed based on Ki-67 expression levels at the wound edge, as determined by performing HRP-DAB IHC staining. The staining results showed that Ki-67 expression was significantly increased in ESWT and HBOT-treated diabetic rats on Day 3 (Table 1; Figure 4B), especially in fibroblasts in the basal epidermis and subcutaneous layer (81.08 ± 3.79 [ESWT] vs. 16.66 ± 1.99 [diabetic control], p < 0.001; and 51.41 ± 6.02 [HBOT] vs. 16.66 ± 1.99 [diabetic control], p < 0.001) and Day 17 after wounding (84.15 ± 3.72 [ESWT] vs. 19.07 ± 4.22 [diabetic control], p < 0.001; and 62.74 ± 5.34 [HBOT] vs. 19.07 ± 4.22 weeks [diabetic control], p < 0.001) compared with the rats in the control group (Table 1; p < 0.001, respectively). Moreover, the expression of Ki-67 at the wound edge of the ESWT group was significantly increased compared with the HBOT group. This finding suggests that ESWT increases cell proliferation earlier and promotes the wound-healing process.

ESWT and HBOT both upregulated eNOS/VEGF expression

Angiogenic effects determined by the expression of VEGF and eNOS in periwound tissue were investigated by using IHC staining. The ESWT and HBOT-treated diabetic groups had increased levels of VEGF and eNOS, especially in fibroblasts and endothelial cells, on Day 10 or Day 17 after wounding compared with the diabetic control group (Table 1; Figure 5). Furthermore, the ESWT group had significantly increased expression levels of VEGF and eNOS at the wound edge twice on Day 10 and Day 17 after wounding compared to the HBOT group. These results indicated that the ESWT treatment group with the best course of treatment showed enhanced angiogenesis compared with the HBOT group.

Table 1 summarizes the IHC staining of the average percentage of positively labeled cells in the periwounding margin. These results indicate that ESWT is more effective than HBOT and the control in accelerating cell proliferation, decreasing oxidative stress, and increasing angiogenesis, resulting in enhanced diabetic wound healing.

DISCUSSION

Unhealed chronic wounds are described by delayed times of inflammation, deferred cell multiplication, poor epithelial recovery, as well as disabled angiogenesis.^1,28 Because of weakened wound healing in diabetic patients, past investigations have detailed different modalities that have had disputable outcomes on wound healing in diabetic patients. In this manner, investigators attempt to plan compelling mediations that can accelerate the process of wound healing. Studies have revealed that ESWT and HBOT both have a positive effect on accelerating wound healing.^10,13,28,29 Our former study demonstrated that ESWT could enhance wound healing and salvage ischemic skin flap tissue survival through increasing topical perfusion, anti-inflammatory responses, and neo-angiogenesis in a rat diabetes model.²⁰ Huang et al.⁷ have shown that ESWT as adjuvant therapy is superior to HBOT to promote DFU healing. Our clinical pilot study also revealed that patients with ESWT treatments have a better outcome than HBOT in enhancing the healing of DFU.²¹ The therapeutic benefits of ESWT and HBOT were attributed to the improvements in topical blood perfusion and cell activity. However, the molecular mechanisms between ESWT and HBOT in diabetic wound healing are still unclear.

In the present study, we compared the effectiveness of ESWT and HBOT in a rodent model of diabetes and investigated the underlying biomechanisms in enhancing diabetic wound healing. Our results revealed that ESWT and HBOT both accelerated diabetic wound healing compared with the control. Moreover, the wound area was significantly reduced in the rats of the ESWT group compared with those of the HBOT group. This result demonstrated that ESWT has a stronger effect than HBOT in facilitating the wound healing process.

Studies have revealed that leukocyte-mediated inflammation is a critical variable in the process of wound healing.²⁷ Histological investigation of the wound region revealed lessened penetration of inflammatory cells on Day 3 after the last session of ESWT and HBOT compared with controls. However, there was no significant difference in leukocyte infiltration at Day 17 after wounding between ESWT or HBOT groups compared with controls. IHC staining showed that compared with the untreated DM control group, the ESWT- and HBOT-treated groups had significantly reduced CD45⁺ expression early and late posttreatment. These results suggest that ESWT and HBOT promote wound healing and are associated with the suppression of early local pro-inflammatory responses. Studies have shown that oxygen free radicals in diabetic rats are significantly increased.³⁰ The expression of 8-OHdG secreted from inflammatory cells, a key oxidative stress biomarker, was also significantly increased in diabetic subendothelial and intima compared to normal blood vessels.²¹ To analyze oxidative changes in chronic wounds, we performed IHC staining for 8-OHdG. The results showed that 8-OHdG levels were significantly reduced in ESWT and HBOT-treated groups during diabetic wound healing. In addition, 8-OHdG expression was significantly decreased in the ESWT group compared with the HBOT group. Further studies are needed to provide more semiquantitative data analysis, such as carbonylated protein, tumor necrosis factor receptor-alpha, or interleukin-1 alpha expression, to elucidate the inflammation-related pathways by which ESWT or HBOT promote wound healing.

In contrast, cellular proliferation and tissue regeneration are important factors for enhancing wound healing. In this experimental study, cell proliferation was analyzed based on Ki-67 expression at the wound edge.²⁶ The results showed that Ki-67 expression was significantly increased in ESWT and HBOT-treated groups, especially in fibroblasts and basal epidermal layers. In addition, the expression of Ki-67 at the wound edge was significantly increased in rats with two sessions of ESWT compared with the rats in the HBOT group (81.08 ± 3.79 [ESWT] vs. 51.41 ± 6.02 [HBOT], p < 0.001 at Day 3; and 84.15 ± 3.72 [ESWT] vs. 62.74 ± 5.34 [HBOT], p < 0.001 at Day 17 after wounding). The results revealed cell migration was increased after HBOT and ESWT treatments. Expression of Ki-67 revealed superiority in ESWT as compared to that in the HBOT group. These findings indicated that ESWT might be at least a part more effective than HBOT in increasing cellular proliferation and facilitating the wound healing process. In this study, we did not detect the subfamily of collagen synthesis after ESWT/HBOT. Further study is still needed to investigate the collagen synthesis-related mechanism and the granulation tissue growth between both groups in detail.

Angiogenic factors assume a significant part in the process of wound healing. In this work, IHC staining was performed to evaluate the expression of VEGF and eNOS at the wound margin posttreatment. The analysis outcomes showed that, compared with the control group, the ESWT and HBOT groups significantly increased the expressions of VEGF and eNOS in the wound edge area on Day 10 and Day 17 after wounding, especially in endothelial cells and fibroblasts. Furthermore, VEGF and eNOS expression levels were elevated in the ESWT group compared with the HBOT group. This result suggests that ESWT is more effective than HBOT in increasing angiogenesis and inducing neovascularization in the wound edge transition zone.

Notwithstanding, this study still has some limitations. This work is restricted by the small number of animals, resulting in relatively low statistical power. Also although this STZ-induced type I diabetic model is common to investigate the wound healing process, most of the clinical cases are type II, diabetes patients. The results of this diabetic wounding model might not be fit for the clinical outcomes. Furthermore, this is a very early study involving comparing the effects of ESWT and HBOT in a rodent chronic wound healing model. Numerous extra examinations are expected to overcome the restrictions of our experimental scheme to clarify unthinking impacts, for example, systemic inflammatory activity detected by flow cytometry or using ELISA, and more per-wound tissue expression related to wound healing molecules, such as inducible nitric oxide synthase, hypoxia-inducible factor 1, matrix metalloproteinase subfamilies, the subtype of collagen synthesis, etc.³¹ Further studies evaluating the systemic effects of ESWT and HBOT are also necessary to better understand these biomechanisms.

In conclusion, the present study demonstrates that ESWT is more effective than HBOT, and the control group in accelerating wound healing. The mechanism should be ESWT to increase more angiogenesis, such as VEGF and eNOS, and promotion of cell proliferation, as well as effects on oxidative damage. This technique represents a feasible method to increase circulation in damaged tissues and diabetic ulcers.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflicts of interest.