**3. Clinical experiences**

Chronic wounds are produced either by an interruption in healing processes, as an effect of lack of positive (vascular supply and neurotrophism) or of an excess of inhibitory factors (metallo proteinases in ECM, some cytokines), or by a lack of switch in inflammatory cell phenotypes, such as in diabetes.

As a final effect, wound bed does not progress beyond detersion, typically presenting itself as necrosis or debris. They are especially present in lower limbs, often as a result of complex mix of the above‐mentioned factors.

Vascular and diabetic ulcers are the most common chronic wounds affecting nearly 2–5% of the general population and have received an important impact in terms of morbidity, absence from work, and social costs.

Traditional wound dressings do not restore vascular supply, which is a *sine qua non* for restarting healing.

An important role is played by vascular surgery and endovascular techniques, which act restoring the lost vascular supply or producing bypasses to revascularize the area.

At present, some novel suggestions come from regenerative surgery.

### **3.1. Lipograft in chronic wounds**

Statistical analysis was conducted with parametric essays for repeated measures (ANOVA) and bonferroni test was used to evaluate intergroup positivity, with a *p* ¼ 0.05 considered as

Our experimental studies have pointed out some important features of antioxidant molecules in impaired wound healing (diabetic mice), as well as the role of some cytokine‐related molecules and endogenous products belonging to natural immunity cascade [2] in normal and

**2.5. Another important contribution to the study of neoangiogenesis and biomaterials**

Our group also developed a collaboration with the group from Padriciano, International Center for Genetic Engineering and Biology, United Nations, to study a model of prefabricated flap in the groin of adult rats creating an artero‐venus loop that was included into a dermal regenerative template; this new regeneration chamber was then injected with different viral vectors (AAV 156) encoding for the production of VEGF. Results were remarkable, demon‐ strating enhancement of neoangiogenesis and neovasculogenesis and the utility of this novel model of regeneration chamber that could act as a bioreactor and stimulate healing and even

Chronic wounds are produced either by an interruption in healing processes, as an effect of lack of positive (vascular supply and neurotrophism) or of an excess of inhibitory factors (metallo proteinases in ECM, some cytokines), or by a lack of switch in inflammatory cell

As a final effect, wound bed does not progress beyond detersion, typically presenting itself as necrosis or debris. They are especially present in lower limbs, often as a result of complex mix

Vascular and diabetic ulcers are the most common chronic wounds affecting nearly 2–5% of the general population and have received an important impact in terms of morbidity, absence

Traditional wound dressings do not restore vascular supply, which is a *sine qua non* for

An important role is played by vascular surgery and endovascular techniques, which act

restoring the lost vascular supply or producing bypasses to revascularize the area.

At present, some novel suggestions come from regenerative surgery.

statistically significant.

**prefabrication**

impaired wound healing [9–13].

158 Wound Healing - New insights into Ancient Challenges

repair poor vascularized tissues [14, 15].

**3. Clinical experiences**

phenotypes, such as in diabetes.

of the above‐mentioned factors.

from work, and social costs.

restarting healing.

The target is endothelial insufficiency, as documented in diabetes and vascular obstructive diseases.

It consists of poor endothelial progenitor cell mobilization and homing, with altered levels of the chemokine stromal‐derived factor‐1 (SDF‐1) at the wound site [2, 6, 16].

The use of tissue engineering techniques such as stem‐cell therapy and gene therapy to improve wound healing has proved a promising strategy [14, 15].

A well‐established clinical experience with lipografting has been obtained in the early years of this century, especially in scar treatment after important fibrotic status, such as in postburn scars and in postradiation mastectomy scars [17, 18].

Since 2001, Zuk et al. documented that lipoaspirate from adipose tissue represents a source of adipose tissue‐derived stem cells, which are adult mesenchymal stem cells [19].

Starting from the first regenerative approach with lipografting on fibrotic tissues, its role has been expanded to chronic wounds, applying as a rationale, and has the potential to induce angiogenesis and regeneration. The potential of ADSCs to differentiate into adipocytes, osteoblasts, chondrocytes, cardiomiocytes, and endothelial cells, in vitro and in vivo, was shown by several authors. In particular, ADSCs are able to express endothelial markers when cultured in the presence of VEGF.

The stimulatory effect of ADSC on cutaneous wound healing may be partially mediated by paracrine effects of ADSCs on other skin cells [20–27].

Application of ADSCs or ADSC‐derived molecules could be an innovative therapeutic approach in the treatment of chronic wounds and other conditions; it has been proposed in association with platelet‐rich plasma [28] or under particular conditions [29].

### *3.1.1. Procedure*

The surgical procedure was performed under local anesthesia together with midazolam medication (see below). The periumbilical area and the hip were the preferred donor site because of the good quantity and quality of dermal fat graft.

With the patient in supine position, the donor area was infiltrated with 250 cc of saline solution (NaCl 0.9%), 0.5 cc adrenalin 1/1000, 10 cc of lidocain 2%, and 10 cc ropivacain 7.5%; the incision to introduce the cannula was made with a no. 11 scalpel (**Figure 1**).

Adipose tissue was harvested through the same incision by a blunt 2 mm cannula connected to a Luer‐Lock syringe of 10 cc, a small amount of aspirate (about 10 cc) was sufficient.

The full syringe was placed into a sterile cup and washed with NaCl 0.9% to remove the anesthetic solution.

The authors used Coleman's technique and centrifuged the fat (3000 rpm for 3 minutes) to separate cellular blood components with infiltration solution, adipocytes with vascular stromal tissue and oil derived from the breakdown of fat cells.

**Figure 1.** Lipoaspirate procedure: ADSC and VASF harvesting, centrifuge and its products on the aspirate, and har‐ vesting of the lipograft to be implanted.

The adipose‐stromal fraction was transferred from a 10 cc syringe to a 1 mL Luer‐Lock syringe to allow a precise control of the amount of injected fat (**Figure 1**).

The adipose tissue fraction was then implanted with gentle care; small "pearls" of adipose tissue were placed at the dermal‐hypodermal junction in the ulcer's edges and into the wound bed. Many radiating passages were made through the same incision, to place fat in different directions.

The access incisions in the donor areas were sutured with Nylon 5/0.

The treated area after surgical procedure was covered with non‐adherent gauze, whereas an elastic adhesive bandage was applied to the fat donor site to prevent hematomas and seromas.

A second grafting session was performed, if needed, 3 months later.

**Figure 2.** Chronic ulcers: before and after treatment with ADSCs.

Four patients were treated, wound closure occurred in approximately 17 days (**Figures 2** and **3**).

**Figure 3.** Lipografting in chronic posttraumatic wound in a diabetic patient. Two sessions were needed to obtain a complete closure.

### **3.2. Mononuclear cells in chronic wounds**

**Figure 1.** Lipoaspirate procedure: ADSC and VASF harvesting, centrifuge and its products on the aspirate, and har‐

The adipose‐stromal fraction was transferred from a 10 cc syringe to a 1 mL Luer‐Lock syringe

The adipose tissue fraction was then implanted with gentle care; small "pearls" of adipose tissue were placed at the dermal‐hypodermal junction in the ulcer's edges and into the wound bed. Many radiating passages were made through the same incision, to place fat in different

The treated area after surgical procedure was covered with non‐adherent gauze, whereas an elastic adhesive bandage was applied to the fat donor site to prevent hematomas and seromas.

to allow a precise control of the amount of injected fat (**Figure 1**).

The access incisions in the donor areas were sutured with Nylon 5/0.

A second grafting session was performed, if needed, 3 months later.

**Figure 2.** Chronic ulcers: before and after treatment with ADSCs.

vesting of the lipograft to be implanted.

160 Wound Healing - New insights into Ancient Challenges

directions.

Cell therapy is an innovative and promising approach for regeneration of damaged tissues. In particular, new scientific evidence shows that the total mononuclears from peripheral blood are cells with high angiogenic and vasculogenic capacity and, in general, in tissue regeneration processes.

Patients with CLI, who suffer from rest pain, nonhealing ischemic ulcers, or necrosis (Fontaine 3–4), rarely respond to standard therapy as drug therapy (e.g., prostaglandin and anticoagu‐ lant, etc.) and surgical revascularization.

The autologous transplantation of peripheral blood mononuclear cells (PBMNCs) can produce tissue regeneration and improve physiological healing process through their paracrine action, consisting in production of cytokines, especially VEGF and bFGF.

The monocells have three principal roles:


The PBMNCs isolated from peripheral blood have the same differentiating and regenerating capacities as the bone marrow mononuclear cells (BMMNCs), but their isolation is simpler and minimally invasive.

Monocytes and macrophages are capable of producing a large variety of growth factors, metalloproteinases, chemokines, and vasoactive substances such as nitric oxide; all can facilitate angiogenesis and arteriogenesis [30–34].

Angiogenesis is characterized by capillary sprouting, endothelial cell migration, proliferation, and luminogenesis to generate new capillaries [14, 32–34].

Arteriogenesis is a positive remodeling of preexisting collateral channels in the limb, as the product of endothelial factors, as well as of infiltrating macrophages [15, 32–34].

During chronic inflammation, macrophages/monocells are polarized in the antimicrobial form (M1), or in the regenerative form (M2). The implantation of concentrated PBMNCs in this condition can address M1 to M2 promoting the regenerative form.

The autologous transplantation of PBMNCs can be considered a valid and safe treatment option for patients with critical wounds [30–34].

### *3.2.1. Procedure*

In the theater, under sedation and local anesthesia of the patient, 120 mL of peripheral venous blood was drawn and added to 12 mL of ACD‐A (anticoagulant by apheresis). This was then processed by the WB Pall Celeris system to obtain 12 mL of concentrated PBMNCs (**Figure 4**).

The concentrated PBMNCs were transferred to a 1 mL Luer‐Lock syringe to allow a precise control during injection (**Figure 4**).

**Figure 4.** Mononuclear cells preparation procedure: 120 mL of peripheral venous are processed by the WB Pall Celeris system to obtain 12 mL of concentrated PBMNCs, and the posterior tibial axis is traced and the injection performed.

After an appropriate surgical cleansing of the wound bed, the concentrate was implanted into the perilesional area in a single‐stage procedure with multiple local subcutaneous perilesional and intralesional injections and intramuscular injections.

The suspension was placed along the relevant damaged vascular axis too, at intervals of 1–2 cm and at a mean depth of 1.5–2 cm, using a 21G needle (**Figure 4**).

After the A‐PBMNCs implant, the wound was always covered with hyaluronic acid monolayer.

This treatment was repeated three times, once a month for three months (**Figure 5**).

**Figure 5.** PBMNC injections were repeated three times, once a month for three months, and a complete healing was registered.

One month after the first treatment, the size of the ulcers of all patients were significantly reduced. At the end of the third session, ulcers seemed totally healed, the skin overlying the wound appeared perfectly normal, and the skin complexion was ruddy (**Figure 5**).

Because of their early capability to stimulate vascular ingrowth, monocell implant can be used in one step only to prepare wound bed to receive an autologous skin graft (**Figure 6**).

**Figure 6.** Monocell implants can be used in one step only to prepare wound bed to receive an autologous skin graft. In this case, a severe necrosis of the midplantar skin was excised and PBMNCs injected. A well‐vascularized granulation tissue was appreciated in 12 days, allowing repair with a split thickness skin graft in this nonweight bearing area.
