Table 2.

Chronic wound models associated with different comorbidities.

Model	Methodology	Utility	Advantages	Disadvantages	References
1. Peripheral artery disease (ischemic) ulcers
1a. Ischemic skin flap and graft models
McFarlane flap	A 10 cm × 4 cm skin flap is formed on the rat’s dorsum. The flap is detached from the surrounding tissues but remains attached at its rear end.	The model is useful for investigating angiogenesis and ischemia-reperfusion. The entire flap can be used as a tissue sample, or it can be sectioned to study the effects of different oxygen levels and biochemical factors.	Easy to implement and reproduce. Multiple tissue samples can be harvested from the flap.	The model does not involve open wounds, but rather induces tissue necrosis, providing a distinct approach from models that focus on inhibited wound healing. Variable patterns of necrosis in the flap, which can differ in extent and severity.	[124]
Flap chamber	Improves the McFarlane flap by introducing a flap chamber that obstructs circulation from basal and lateral sides of the flap tissue.	The model has been employed for investigating angiogenesis and ischemia-reperfusion. The chamber isolates the dorsal skin flap from surrounding skin for more consistent and replicable results.	More stable outcomes compared to the suture model in the McFarlane flap. Linear gradient of ischemic pattern from the distal to the proximal end of the flap.	The model focuses on tissue necrosis rather than inhibited wound healing.	[125]
H-shaped double flap	An 8-cm long H-shaped double flap is created on the back of each animal by making perpendicular cuts through the skin and panniculus carnosus. After elevating the flaps and cutting the central vein’s perforating branches, the flaps are sutured back into position.	The model is effective for examining tissue ischemia with progressively recovering blood flow. Initially, blood perfusion drops to 7 % of its normal rate on the first postoperative day, and then gradually returns to normal by day 16.	No full-thickness skin necrosis on the edges of the flap. Blood flow remained consistent across flaps of varying widths, ranging from 1 to 2.5 cm, showing no significant differences.	No measures were taken to prevent healing of the flap beds.	[126]
Bipedicle flap	A long rectangular flap of skin is created by making incisions along the long sides, while the short sides were left intact, forming a bipedicle flap. The long sides of the raised flap are secured with surgical staples. The full-thickness excisional wounds are made using the biopsy punch close to the flap center.	The model is useful for studying delayed healing of full-thickness wounds, with wounds remaining open for 12 or more days after injury.	Multiple wounds per animal can be induced. Delayed healing of wounds without tissue necrosis.	Reperfusion in the flap is primarily attributed to angiogenesis. No measures were taken to prevent healing of the flap beds.	[127,128]
Bipedicle flap with silicone sheeting	This bipedicle flap model incorporates the use of a silicone sheet placed beneath the flap to prevent reattachment and revascularization from the underlying tissue. Full-thickness wounds are induced at the center of the flap.	The model has been employed in investigating ischemic wound healing. The bipedicle flap remained viable throughout the entire ischemic wound healing period of about 28 days, in contrast to non-ischemic wounds which healed within approximately 10 days	Multiple wounds per animal can be induced. Delayed healing of wounds without tissue necrosis. The use of the silicone sheet not only prevents reperfusion but also minimizes wound contraction.	Reducing the flap size to less than 2.0 cm, or exposing the flap to trauma, can increase the risk of necrosis. Minor variations in environmental factors, such as temperature or stress levels, might cause biochemically detectable variations in the experimental outcomes.	[129]
Skin graft	A square of skin, along with the panniculus carnosus, is excised from the back’s midline. The graft is then sutured back onto the underlying muscle fascia using nonabsorbable stitches.	Pharmacological studies, stem cells therapy, angiogenesis research	A large area of the skin is under low-perfusion conditions. The perfusion restored to 75 % of normal by day 14.	The model is designed for evaluating graft survival and preventing necrosis, not for the formation of a chronic wound.	[130]
1b. Arterial insufficiency PAD models
Sodium laurate-induced limb necrosis	A pharmacological model for PAD. Sodium laurate solution is injected into the right femoral arteries of rats to trigger ischemia.	This model is used to assess to evaluate the effects of long-term drug administration on hindlimb muscle circulation.	Severe injury to the skin tissue. The reperfusion is limited, hindering the restoration of limb tissue functionality.	The proposed method results in necrosis, not ulceration.	[131]
Iliac and femoral artery ligation	A midline laparotomy is performed, ligating all branches from the left side of the aorta beyond the renal arteries and all branches originating from the left iliac artery using resorbable sutures. In a subsequent operation via the left inguinal incision, the femoral artery near the origin of the superficial epigastric artery is ligated.	This model is effective for studying limb ischemia over a prolonged duration. Reperfusion decreased from day 1 to week 4 compared to a control group.	Low mortality No signs of gangrene or limb loss Reduced perfusion in foot skin for up to 8 weeks Normal weight post-surgery.	The model does not involve open wounds.	[69]
Ipsilateral femoral/iliac artery ligation	First, a right iliac artery ligation approximately 5 mm below its bifurcation from the aorta is performed using a 3-0 silk suture. After 3 weeks, the right femoral artery below the branching of the arteria profunda femoris is also ligated.	The model is designed to mimic the impairment of nutritive perfusion observed in patients with PAD.	More sustained disruption of arterial and capillary perfusion than a single iliac artery ligation, which typically restores blood flow within 2–5 weeks.	The model does not involve open wounds.	[65]
Ameroid constrictor	Utilizes a 0.75-mm internal diameter ameroid constrictor around the left common iliac artery and left femoral artery.	The model is aimed at simulating ischemic conditions in PAD by gradually narrowing vessel diameter.	Using of ameroid constrictors results in slower, less comprehensive blood flow recovery, lower dermal blood flow in the feet, and reduced oxygen tension in the gastrocnemius tissue. Following the placement of ameroid constrictors, blood flow in rats recovers to around 66 % by the 40th day post-operation, whereas in rats with artery ligation, recovery reaches approximately 60 % within the first two weeks.	The model does not involve open wounds. Critical limb ischemia is induced through acute arterial occlusion, rather than gradual arterial occlusion, as determined by the clinical ischemia index.	[70]
Apolipoprotein deficiency/ligations/ameroid constrictor	A modified, three-stage method for inducing chronic hindlimb ischemia and peripheral artery disease (PAD) in mice. Stage 1: Use of apolipoprotein E-deficient (apoE−/−) animals. Stage 2: Ameroid constrictor applied to the femoral artery. Stage 3: 14 days later, ligation of the femoral artery and its side branches near the inguinal ligament and popliteal bifurcation.	The model is designed for studying severe, prolonged ischemia in older mice (20 months), providing insights into long-term ischemic effects without natural recovery.	Mice subjected to three-stage ischemia exhibit more pronounced ischemia and impaired limb usage compared to two-stage ischemia. Diminished treadmill performance in mice undergoing three-stage ischemia, mirroring PAD symptoms in patients. Old mice are used (20 months).	The model does not involve open wounds. Mice are used instead of rats.	[71]
Lower limb artery removal	Multiple ligations are performed on the following arteries: common femoral artery, common external iliac, proximal common femoral, external iliac and saphenous arteries at knee level, common iliac and proximal common femoral arteries, common iliac and common femoral artery at knee level. Subsequent arterial resection involves complete excision from the proximal common femoral to the saphenous artery, external iliac to the saphenous artery, and common iliac to the saphenous artery. Bilaterally full thickness wounds (1.2 × 0.8 cm) were created on the dorsal of the feet of the rats.	The model is useful to assess the impact of ischemia on open wound healing. The foot dorsum was chosen for wounding due to its distal location relative to the arterial lesion, its fixed and flat surface, and its similarity to human condition.	In the ischemic limb, blood flow dramatically drops to 9.3 % of the control limb’s level immediately post-insult and stabilizes to about 85 % after 34 days. The study duration was adequately long (40 days).	This model does not represent the gradual formation of the PAD ulcer resulting from arterial insufficiency.	[72]
2. Diabetic wounds
McFarlane flap model	STZ is injected intraperitoneally into rats. After several days, animals exhibiting elevated blood glucose levels are identified as diabetic. Subsequently, a McFarlane flap is surgically created on the backs of these diabetic rats.	This model is employed to assess the effectiveness of drugs in mitigating distal necrosis of flaps in diabetic rats.	Easy to implement and reproduce. Multiple tissue samples can be harvested from the flap.	Lack of using high fat diet in inducing diabetic model. Ischemic reperfusion injury because of vasodilation (24 h after creating McFarlane flap) High mortality rate (13 out of 48).	[87]
Skin graft	Diabetes is induced through an injection of STZ. A square skin section, including the panniculus carnosus, is surgically removed from the dorsal midline. The panniculus carnosus is then carefully excised to create a full-thickness skin graft, which is subsequently reattached to the underlying muscle fascia.	The model is designed for evaluating graft survival and preventing necrosis.	Easy to implement and reproduce. Multiple tissue samples can be harvested from the graft.	The model is not designed to simulate the gradual formation of chronic wounds.	[132]
Ischemic Foot Model	Hyperglycemia is induced in rats using STZ injection, followed by the resection of the external iliac, femoral, and saphenous arteries. Through a shaved longitudinal incision in the inguinal region, the external iliac and femoral arteries are dissected from the common iliac to the saphenous arteries.	The model is effective for researching critical limb ischemia.	The model shows a direct correlation between hyperglycemia and acute ischemic necrosis.	The model induces tissue necrosis without creating open wounds. High dropout rate: 71 % of diabetic rats developed total necrosis	[61]
Excisional diabetic wound model	Diabetes was induced by intraperitoneal STZ injection. A full-thickness excision wound, spanning an area of 200 mm2 and 0.2 cm in depth, was created on the dorsum using a surgical blade and pointed scissors.	The model is used for assessing the efficacy of a topical therapy on diabetic wounds.	Substantial wound size. The study duration was adequately long (21 days).	Wound closure by contraction is not inhibited.	[133]
3. Venous stasis ulcers
Vein obstruction	The model includes two phases: Acute: The common femoral vein is bilaterally ligated with cotton ties. Following a laparotomy, the common iliac veins are ligated bilaterally. Chronic: After repeating the acute phase, all incisions were closed with two layers of absorbable sutures.	The model is effective to study an intercellular adhesion molecule in venous disease.	Simple cotton thread ligations created an acute hypertension model, while two-layer ligations with absorbable sutures led to a chronic CVI condition. An 11-fold increase in hind limb vein pressure compared to the forelimb in the control group was recorded on day 14 post-ligation.	The model does not involve the creation of open wounds. Short duration of the study (14 days).	[90]
Arteriovenous fistula	An incision is made on the femoral artery (0.5 mm) to induce an arteriovenous fistula, followed by the creation of a femoral vein ligation above the fistula.	The model is useful to examine the inflammatory process in valve remodeling associated with acute and chronic venous hypertension.	Increased blood pressure in the femoral vein distal to the fistula of 96 ± 9 mmHg. Allow to observe the time-dependent disappearance of the venous valve in the fistula model. A proper time frame of 42 days was set for the induction of venous diseases.	The model does not involve the creation of open wounds.	[134]
Vertical position/gravity model	Rats are placed in head-up and head-down positions using tube-like plastic cages that support the rat’s body in a tilted position.	The model aims to investigate physiological responses to gravitational loading or unloading of extremity veins.	Induction of the venous stasis model mimicking precisely the human etiology. Long-term duration of the study	The model does not involve the creation of open wounds.	[93,135]
Occlusion and gravity	This model combines gravity and the occlusion of the saphenous vein. A plastic clip was applied to partially occlude the saphenous vein just below its confluence with the deep femoral vein. For the final four weeks prior to sacrifice, rats were housed in tube-like cages inclined at a 45-degree angle in a head-up position.	The model aims to investigate physiological responses to gravitational loading or unloading of extremity veins.	Remodeling of the vein is observed, including local dilations and convoluted courses, like human morphologies. Long-term duration of the study (12 weeks).	The model does not involve the creation of open wounds.	[94]
Skin flap and venous congestion model	An abdominal skin flap measuring 3 × 6 cm, supplied by the epigastric artery and vein, is elevated and subjected to a 2-h occlusion of both the epigastric artery and vein. Following a period of 24 h, a secondary, 5-h occlusion is specifically applied to the vein. During the secondary ischemia phase, the flaps were resutured. After the 5-h period of secondary ischemia, the venous clamp was removed.	The model aims to investigate techniques for relieving venous congestion through targeted fluid transport interventions.	Large area of the abdominal skin flap The mean perfusion value in the flaps remained constant, maintaining a level of 15 % ± 10 % on day 7.	The model did not display all the typical clinical signs of venous congestion. By day 7, 80 % of the flaps become fully necrotic.	[95]
4. Pressure ulcers (PU)
Weight-induced pressure	A steel plate is inserted into the rat’s peritoneal cavity, and constant pressure is applied to the abdominal wall using metal ingots for a duration of 2–4 h before being removed.	This model is employed to investigate deep tissue injury markers by analyzing exudate.	The model yields a significant amount of exudate. Muscle necrosis can be observed.	The model does not result in the development of open ulcers. The short duration of the study may not be suitable for simulating chronic wound formation.	[101,136]
Piston-induced external pressure	A flat plastic piston with an elastic spring is used to deliver constant, quasi-static pressure to the gracilis muscle in proximal limbs. Two different experimental approaches are used. In long-term experiments (lasting 120, 240, or 360 min), pressure is exerted externally on the skin to reduce the risk of cell death from infection. For shorter durations (up to 80 min), the pressure is directly applied to the gracilis muscle following skin retraction.	Study of muscle tissue damage in rats subjected to different pressure magnitudes and durations.	The piston ensures precise and repeatable tissue compression.	The model does not result in the development of open ulcers. The short duration of the study may not be suitable for simulating chronic wound formation.	[25]
Magnet-induced pressure	The model involves implanting a neodymium magnet in the peritoneal cavity after a transverse lower abdominal incision. A stronger neodymium magnet is then placed on the skin. The pressure was applied for 2–4 h and then removed, followed by monitoring the rats for up to one week.	This model is designed to study the pathogenesis of pressure ulcers.	Severe edema in the skin and subcutaneous tissue is achieved. Easy to control the time and magnitude of pressure.	The model does not result in the development of open ulcers. The short duration of the study may not be suitable for simulating chronic wound formation.	[101]
Cyclic magnet compression	A sterile steel disk (15 mm diameter, 0.3 mm thickness) is placed under the muscle through a 2 cm incision, which was then sutured. On post-operative day 3, a magnetic disk (15 mm diameter, 1500 G) is placed externally on the skin. Two hours after the initial pressurization, the magnets were removed for 30 min to allow for ischemia reperfusion. This pressurization cycle was repeated 5 times a day.	The model is utilized to simulate the development of stage II pressure ulcers.	Stage II pressure ulcers develop after 2–3 days of cyclic pressure application.	The model does not simulate higher degrees of bedsores. Compressing the skin on both sides neglects the role of underlying tissue in ulcer formation.	[103]
Prolonged magnet compression	This model is an adaptation of the previous one, extending the duration to up to 6 days. A steel plate is inserted into the rat’s back muscle. A magnet was positioned externally on the skin for 2 h, repeated 5 times daily over 6 days, to simulate a deep tissue injury.	The model is designed to investigate the impact of secondary factors (in particular, smoking) on the wound healing process of stage 4 pressure ulcers.	Stage IV pressure ulcers develop on day 6 of cyclic pressure application.	Compressing the skin on both sides neglects the role of underlying tissue in ulcer formation.	[108]
Deep tissue injury at bony prominences	A hemispherical implant is placed on and beneath the lateral surface of the tibia, affecting the tibialis anterior muscle. Four weeks post-implantation, pressure is applied to the tissue above the implant for 24 h.	These models simulate the development of pressure ulcers when constant pressure applied on bony prominences.	The clinical relevance is enhanced by mimicking the effect of bony prominences on DTI. Formation of necrotic lesions within the muscle tissue.	This model does not result in the development of open wounds.	[105]
Spinal cord injury	In the SCI model, a complete transverse spinal cord cut at T10, along with a laminectomy at T9, is performed to induce SCI. Six weeks post SCI induction, pressure is applied to the skin surface over the midpoint of the tibialis anterior muscle belly using a custom-made device for 24 h.	These models simulate the development of pressure ulcers under chronic SCI condition.	Clinical relevance is enhanced by incorporating chronic SCI. Formation of necrotic lesions within the muscle tissue.	This model does not result in the development of open wounds.	[105]
5. In vivo biofilm models
Infected full thickness wound model	A 6 mm diameter full-thickness wound is created on rats using a biopsy punch and inoculated with Pseudomonas aeruginosa strain PAO1. Wounds are harvested at 8 h, and 1-, 3-, and 7-days post-wounding.	The model tests the contribution of biofilm in delaying wound healing.	Easy setup, Biofilms form by day 1 post-wounding. Use of fluorescent protein plasmid for biofilm tracing	Short study duration. Mono-species bacterial model. Wound closure by contraction is not inhibited. Small size of the wound.	[121]
Infected full thickness wound model	Round 2-cm diameter full-thickness wounds are created on rat flanks with sterile scissors. Two bacterial concentrations are used for colonization and infection groups, respectively. Wounds are harvested on day 6 post-wounding.	The model evaluates host-pathogen relationships affecting wound healing.	Substantial wound size. Comparative study with varying bacterial concentrations. Use of both young (7 weeks) and old (6 months) rats.	Short study duration. Mono-species bacterial model. Wound closure by contraction is not inhibited.	[123]
Modified Walker-Mason model for full-thickness scald burns	A full-thickness burn is inflicted on male rats using an insulated mold with an opening, which exposes approximately 10 % of the total body surface area, to 99 °C water for 6 s.” Wounds are inoculated with P. aeruginosa, applying two distinct concentrations for different animal groups. Tissues are harvested on days 1, 3, 7, and 11 post-wounding.	The model is designed to assess the healing process of contaminated full-thickness scald burns.	Substantial wound size. Strong biofilm infections form in full thickness burn wounds. Peak bacterial load is reached within 7 days.	Short study duration. Mono-species bacterial model. Wound closure by contraction is not inhibited.	[137]
Modified Walker-Mason model for partial-thickness scald burns	Partial-thickness burn covering 10 % of total body surface area is induced by immersing in 99 °C water for 3 and 6 s, respectively. Three bacterial inoculum sizes are applied for different animal groups.	The model is designed to assess the healing process of contaminated partial-thickness scald burns.	Substantial wound size. Biofilms form within 7 days post-burn.	Short study duration. Mono-species bacterial model. Wound closure by contraction is not inhibited.	[138,139]
Infected pressure wound	To induce pressure ulcers in rats, incisions is made on the lateroabdominal region, and a metal plate is inserted subperitoneally. Ischemic wounds are produced by applying an indenter for 3 h. P. aeruginosa strains PAO-1, PAO-MW1, and PAO1- MP3 are used for inoculation. Skin samples were harvested on day 3.	The model is developed to study the infected pressure ulcers.	Substantial wound size. Multi-species bacterial model Tracking the bacteria by plasmid.	Short study duration.	[140]
Infected diabetic wound	Diabetes was induced by intraperitoneal STZ injection. A punch biopsy instrument is used to create a full-thickness, round wound through the panniculus carnosus in the interscapular region on the upper back of each rat. The skin flap is then excised using iris scissors. An oval-shaped silicone splint is affixed to the skin using immediate-bonding cyanoacrylate and reinforced with interrupted nylon sutures to secure its position. These wounds are subsequently inoculated with bacterial suspensions of either Staphylococcus aureus or Pseudomonas aeruginosa.The animals are sacrificed on day 11.	The model is used for evaluation of topical antimicrobial therapy in diabetic wound infections.	Substantial wound size. Multi-species bacterial model. Wound splinting to prevent contraction. Mid-study wound debridement to evaluate contamination without sacrificing the animals.	The model does not simulate the gradual development of diabetic ulcers.	[141]