Debriding agents

• •


#### Other options -Prp, PDGF,

low-frequency ultrasound therapy (MIST, celleration) with composite dressings, and synthetic skin substitutes

Types of dressing Negative pressure wound therapy

(NPWT)

• Velcro adjustable

• Larval Near-infrared

Dehydrated

chorion membrane

•

PURION

Dehydrated

allograft (dHUC)

• Honey dressing (e.g., Trauma wounds

•

Polymeric membrane

ings (PMD)

> •

Table 2. Types of dressings and patches that are currently available.

PMD + silver

 dress-

the ED

Standard protocol of care in

•

Resorbs blister exudate,

reduces edema

> •

Silver–Antibacterial

http://dx.doi.org/10.5772/intechopen.82614

207

impregnated

 absorbent

Contains scoparium honey

Leptospermum

• •

Moderate to heavy exudative

wounds

Maintains a moist

environment

MANUKAhd)

PURION PLUS process

 human umbilical cord

 (dHACM)

 human amnion/

spectroscopy

 (NIRS)

debridement

 therapy

Ready wrap

 wraps

Pico

Primary or secondary

Functions/indications

Pros

Cons

Frequency

dressing

change

Changed every

72 h

 of

dressing

Computerized

device applies negative

pressure which decreases

wound healing time

Limitations–training

patient and families to apply

correctly with

Limitations

of the larva mortality, escape

 are

asphyxiation

Offloading

region provided the best chance for

larval survival Chronic wounds regenerate

damaged tissue

•

Chronic wounds in difficult-to-

heal areas

> •

Provides connective

matrix to supplement

tissue

 tissue

 damaged

Facilitation of Wound Healing Following Laparoscopic and Conventional Abdominal Surgery…

 of the

coccyx/sacral

 the

 vacuum


Types of dressing

Hydrocolloids

Primary or secondary

•

Used in epithelizing ulation wounds Ulcers with light to moderate exudate [9]

> •

Gel-forming

moisture and protects wound

> •

Hydrocolloid

lytic wound exudate in contact with

necrotic tissue (slough and

eschar)

•

Paste and powders increase

absorptive capacity

> •

•

Highly absorbent of exudate

which keeps it off the wound

and decreases damage to sur-

rounding tissue

> •

Deep cavity wounds to prevent

premature closure, absorbing

exudate and maintaining

moist

> •

•

Debriding

• • Other options


ultrasound

celleration)

dressings, and synthetic skin

substitutes

 with composite

 therapy (MIST,

low-frequency

Accuzyme (Health point)

Santyl (Health point)

 agents

Diabetic foot ulcers [10]

Weeping ulcers

environment

 a

Ulcers highly exudate

•

Absorbent char-

•

Require sec-

• •

Silver-Up to 7

Varies

ondary dress-

ing usually

acteristics

•

Comfortable

and form well to

•

Drying effect

days

wound

•

Used for painful

/risk for shear

injury pressure

ulcers

Foams

Primary

> •

• •

Foam with AMD

PolyMem

Generic foam

debridement

 by keeping

 sheets help auto-

 covering keeps in

 and gran-

•

Easy applica-

•

Never used

Every 7 days

206 Wound Healing - Current Perspectives

for infected

wounds

tion, forms to

wound well decreasing pain

•

Not used for

moderate to heavy exuda-

tive wounds

•

Used to protect

body areas at

risk for friction

injury or tape

•

Reside and foul odor may

arise from breakdown

product

 of

injury

• •

Duoderm

Generic

hydrocolloids

Primary or secondary

Functions/indications

Pros

Cons

Frequency

dressing

change

 of

dressing


surgery being performed, yet the umbilicus is nearly always used as a port site to allow the camera to pass through at this time; the technique is still under development, and further improvement such as artificial intelligence (AI) or real-time, dynamic AI system will speed up the procedure, enhance safety, and improve outcomes. Once the operation is over, surgical excisions can be closed by sutures, staples, steri-strips, tissue glue, or a combination of these agents. The wound should be covered in a protective dressing like gauze and attached with a paper adhesive tape and kept dry for a few days, before normal washing can resume. Pre- and postoperative prophylactic antibiotics can be administered for laparoscopic surgery but are

Facilitation of Wound Healing Following Laparoscopic and Conventional Abdominal Surgery…

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209

Wound care for an incision starts before making the initial cut and lasts until the end of the patients' healing process; it's important to maintain proper surgical field cleanliness and to prepare a patient with the right technique before performing any type of laparoscopic surgery [5]. Medical patients that are undergoing laparoscopic surgery should be given antibiotics usually a first-generation cephalosporin 30 minutes prior to the incision as well as prepped with betadine, a povidone iodine, or hexachlorophene before surgery. Giving antibiotics postoperatively depends on the type of the laparoscopic procedure done; the patient is normally given 1–3 postoperative doses, and if it's a colorectal surgery, then three doses is usually given

When performing a laparoscopic surgery, various access approaches have been used. For the removal of the gallbladder or appendix, all the instruments may be inserted at a single incision using a Gel POINT (Applied Medical), SILSPORT (Medtronic), or Triport (Olympus) access platform. This approach is especially appropriate in a patient who is young and thin. It is more acceptable in female patients as it yields a great esthetic result, allows a wide range of motion during the surgery, and is a minimally invasive method [6, 7]. It is important to ensure if there is any suspicion of contamination or infection such as appendicitis, cholecystitis, and cystectomy that an end catch bag is used, to minimize the chance for an infection. A specimen retrieval bag, such as endocatch or endobag (Covidien), reliacatch (Medtronic), endopouch (Ethicon), and Conmed (eSutures), is used which avoids spillage and contamination of the infected specimen to minimize the chance for an infection. When closing a laparoscopic incision, there are options of using subcuticular stiches or staples. If the wound is small, a tissue adhesive skin glue such as Dermabond can be used to close the skin. The incision is then covered using Telfa, a nonadherent dressing gauze which conforms to the wound and absorbs light drainage; the gauze is then held in place with Tegaderm. Tegaderm is a semiocclusive transparent film, which self-adheres and allows insensible water loss and prevents the entry of bacteria and proteins; transparent films are known to have the fastest healing rates and lowest infection and are the most cost-effective [5]. The initial postoperative dressing can be removed in 48 h if the wound remains dry. No matter whether traditional or minimally invasive approach is used, surgical complications can occur after surgery. The most common complica-

tions include wound, infections, and dehiscence. Their management is presented here.

debatable and not recommended by the WHO if the wound is not contaminated.

4. Routine care postsurgery

due to the higher risk for infection.

Table 3. Future options for wound care.

intraoperative findings or complications. Historically, the earliest surgeries were crude and were performed out of desperation. It wasn't until the 1900s that the risk of dying after surgery mainly due to infection was less than 50%. After the turn of the century, the likelihood of surviving surgery was greater than the chance of dying during or immediately after surgery. Early techniques were rudimentary, or even barbaric by today's standards, as anesthesia was not commonly used until the mid-to-late 1800s.

Today, surgery takes a wide variety of forms and is often performed using minimally invasive techniques. This has shortened recovery times, improved outcomes, and minimized complications for most patients. However, laparotomy is still used in some types of surgery, especially in organ transplantation (such as the liver, pancreas, and kidney), because it allows extensive dissection and anastomoses.

For traditional abdominal surgery, the incision should interfere minimally with abdominal wall function by preserving important abdominal structures and heal with adequate strength to reduce the risk of wound disruption and herniation. During surgery, wisely chosen incisions and correct methods of making and closing such wounds are factors of great importance. Any mistake, such as a badly placed incision, inept methods of suturing, or random selection of suture material, may result in serious complications such as unnecessary functional disruption, hematoma formation, ugly scar formation, wound dehiscence and herniation, or complete disruption of the wound [3].

The specific surgical incision will depend on the underlying pathology, site, patient factors, and the surgeon's preference and experience. The key principles of making surgical incisions are (1) for maximal wound strength with minimal scarring, incisions should try to follow Langer's lines where possible, and (2) where possible, muscles should be split and not cut. There are several types of laparotomy, such as longitudinal (vertical), transverse, and oblique, and each has various sizes and positions to fit different surgical goals.

### 3. Minimally invasive surgery

The recent development of endoscopic and laparoscopic technology has revolutionized traditional surgery concepts facilitating patient friendly access to even the most remote of abdominal organs [4]. Laparoscopic surgery requires small incisions to be made in the skin, which allows instruments to be passed into the abdominal cavity. Common instruments include the camera, cutting and dissecting scissors, and grippers. The port sites will vary depending on the surgery being performed, yet the umbilicus is nearly always used as a port site to allow the camera to pass through at this time; the technique is still under development, and further improvement such as artificial intelligence (AI) or real-time, dynamic AI system will speed up the procedure, enhance safety, and improve outcomes. Once the operation is over, surgical excisions can be closed by sutures, staples, steri-strips, tissue glue, or a combination of these agents. The wound should be covered in a protective dressing like gauze and attached with a paper adhesive tape and kept dry for a few days, before normal washing can resume. Pre- and postoperative prophylactic antibiotics can be administered for laparoscopic surgery but are debatable and not recommended by the WHO if the wound is not contaminated.

### 4. Routine care postsurgery

intraoperative findings or complications. Historically, the earliest surgeries were crude and were performed out of desperation. It wasn't until the 1900s that the risk of dying after surgery mainly due to infection was less than 50%. After the turn of the century, the likelihood of surviving surgery was greater than the chance of dying during or immediately after surgery. Early techniques were rudimentary, or even barbaric by today's standards, as anesthesia was

Today, surgery takes a wide variety of forms and is often performed using minimally invasive techniques. This has shortened recovery times, improved outcomes, and minimized complications for most patients. However, laparotomy is still used in some types of surgery, especially in organ transplantation (such as the liver, pancreas, and kidney), because it allows extensive

For traditional abdominal surgery, the incision should interfere minimally with abdominal wall function by preserving important abdominal structures and heal with adequate strength to reduce the risk of wound disruption and herniation. During surgery, wisely chosen incisions and correct methods of making and closing such wounds are factors of great importance. Any mistake, such as a badly placed incision, inept methods of suturing, or random selection of suture material, may result in serious complications such as unnecessary functional disruption, hematoma formation, ugly scar formation, wound dehiscence and herniation, or complete

The specific surgical incision will depend on the underlying pathology, site, patient factors, and the surgeon's preference and experience. The key principles of making surgical incisions are (1) for maximal wound strength with minimal scarring, incisions should try to follow Langer's lines where possible, and (2) where possible, muscles should be split and not cut. There are several types of laparotomy, such as longitudinal (vertical), transverse, and oblique,

The recent development of endoscopic and laparoscopic technology has revolutionized traditional surgery concepts facilitating patient friendly access to even the most remote of abdominal organs [4]. Laparoscopic surgery requires small incisions to be made in the skin, which allows instruments to be passed into the abdominal cavity. Common instruments include the camera, cutting and dissecting scissors, and grippers. The port sites will vary depending on the

and each has various sizes and positions to fit different surgical goals.

not commonly used until the mid-to-late 1800s.

dissection and anastomoses.

Table 3. Future options for wound care.

208 Wound Healing - Current Perspectives

disruption of the wound [3].

3. Minimally invasive surgery

Wound care for an incision starts before making the initial cut and lasts until the end of the patients' healing process; it's important to maintain proper surgical field cleanliness and to prepare a patient with the right technique before performing any type of laparoscopic surgery [5]. Medical patients that are undergoing laparoscopic surgery should be given antibiotics usually a first-generation cephalosporin 30 minutes prior to the incision as well as prepped with betadine, a povidone iodine, or hexachlorophene before surgery. Giving antibiotics postoperatively depends on the type of the laparoscopic procedure done; the patient is normally given 1–3 postoperative doses, and if it's a colorectal surgery, then three doses is usually given due to the higher risk for infection.

When performing a laparoscopic surgery, various access approaches have been used. For the removal of the gallbladder or appendix, all the instruments may be inserted at a single incision using a Gel POINT (Applied Medical), SILSPORT (Medtronic), or Triport (Olympus) access platform. This approach is especially appropriate in a patient who is young and thin. It is more acceptable in female patients as it yields a great esthetic result, allows a wide range of motion during the surgery, and is a minimally invasive method [6, 7]. It is important to ensure if there is any suspicion of contamination or infection such as appendicitis, cholecystitis, and cystectomy that an end catch bag is used, to minimize the chance for an infection. A specimen retrieval bag, such as endocatch or endobag (Covidien), reliacatch (Medtronic), endopouch (Ethicon), and Conmed (eSutures), is used which avoids spillage and contamination of the infected specimen to minimize the chance for an infection. When closing a laparoscopic incision, there are options of using subcuticular stiches or staples. If the wound is small, a tissue adhesive skin glue such as Dermabond can be used to close the skin. The incision is then covered using Telfa, a nonadherent dressing gauze which conforms to the wound and absorbs light drainage; the gauze is then held in place with Tegaderm. Tegaderm is a semiocclusive transparent film, which self-adheres and allows insensible water loss and prevents the entry of bacteria and proteins; transparent films are known to have the fastest healing rates and lowest infection and are the most cost-effective [5]. The initial postoperative dressing can be removed in 48 h if the wound remains dry. No matter whether traditional or minimally invasive approach is used, surgical complications can occur after surgery. The most common complications include wound, infections, and dehiscence. Their management is presented here.

### 5. Postoperative wound infections

Surgical site infections are defined as infections that occur 30 days after surgery with no implant or within 1 year if an implant is placed and infection appears to be related to surgery. Surgical site infections (SSI) are seen in about 4% of clean wounds and 35% of contaminated wounds, so they are generally rare. However, certain risk factors predispose a patient to an SSI, which include diabetes, obesity, immunosuppression, cardiovascular disease, smoking, cancer, preventative surgery, malnutrition, and prior irradiation [8]. Obese patients are at increased risk especially if the incision is in the umbilical area due to fatty tissue not being well vascularized, the difficulty of cleaning it, and usually multiple incisions being made in obese patients. SSIs are associated with substantial morbidity and mortality. Patients with SSI are twice as likely to die, 60% more likely to be admitted to the intensive care unit, and more than five times more likely to be readmitted to the hospital after discharge.

depends on where the incision site is. The subcuticular superficial arteries can bleed or the inferior epigastric vessel if there was a lateral incision made. Usually the nurse will inform the doctor that the dressings are saturated and a pressure dressing is placed until the bleeding

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211

Some other complications from surgery include seromas, hematomas, fascial dehiscence, hernias, evisceration, and nerve injury. Seromas and hematomas are not common with laparoscopic incisions and seen more with open surgery; when a seroma or hematoma has occurred, it can be aspirated, and if a seroma recurs, it should be aspirated until it's fully gone. The wound can also have dehiscence; the main reasons for wound dehiscence are failure of the suture to remain anchored to the fascia, suture damage, knot failure, and stiches applied too close together. In 95% of abdominal wall dehiscence, the sutures and knots are intact, but the suture has pulled through the necrotic fascia due to the sutures being too close to the edge of a wound or under too much tension. If fascial disruption is suspected, wound exploration should be done in the operating room; complete dehiscence is a surgical emergency and is associated with a 10% mortality rate [4]. Management of SSI depends on the severity of the infection. Minor and superficial infections may be treated with antibiotic therapy. If the infection is deep and severe with pus or fluid drainage, the wound should be opened, explored, drained, irrigated, debrided, and dressed open, and the dressing should be changed daily or more often if the drainage is severe. When the infection has cleared and there is granulation tissue growth, the wound can be closed by secondary intention [8]. Antibiotic therapy is only used for wound infections associated with cellulitis or edema. Superficial incisional infections that have been opened can usually be man-

The quality of postoperative wound healing plays a significant role and is critical in patient recovery and rehabilitation because mortality and morbidity rates are affected by surgical wound dehiscence (SWD). SWD is defined as partial or total disruption of any or all layers of operative wounds—from simple skin dehiscence and hernia formation to the most severe and potentially lethal forms characterized by evisceration, gastrointestinal anastomotic leaks, pancreatic fistulas, and vascular pseudoaneurysms. The impact of SWD can be considerable: increased mortality, delayed hospital discharge, readmission, future surgery, delayed adjuvant treatment, suboptimal esthetic outcome, and impaired psychosocial well-being. SWD occurrence rates can vary significantly, from 0.65 to 2.1% in sternotomy to as high as 16.9–41.8% in pilonidal sinus surgery, and the cost for these wounds was \$13.1 billion according to Medicare

Bleeding is a relatively rare complication if the wound is inspected carefully before closing. Bleeding usually happens early; the trocar can tamponade during the surgery and can injure a

aged without antibiotics if there is no associated cellulitis [5].

6. Wound dehiscence

data from a 2014 report.

7. Bleeding and hematoma

stops.

Signs and symptoms of incisional infection vary significantly depending on the types, severity, and pathogens. The most frequent symptoms include fever, feeling of malaise, fluid drainage, increased wound pain, redness and swelling around the wound, and loss of function and movement. SSI may be caused either by endogenous or exogenous microorganisms. Most SSIs are caused by endogenous microorganisms present on the patient's skin at the site of surgical incision, and Gram-positive bacteria such as Staphylococcus aureus are the most common microorganisms. SSI may also be caused by organisms within the patient's body that are exposed during surgery, such as Gram-negative microorganisms in the gastrointestinal tract. Exogenous sources of microorganisms include surgical instruments, operating room surfaces, the air, and personnel. Usually all wound infections happen on day 5 after surgery, and it's safe to suspect a staph infection due to the commonality of it. Of the rarer types of infections, Group A strep is seen on day 2 and clostridium usually is seen on day 3. To diagnose surgical site infections, a clinician should look for redness, induration, warmth, pain, purulent wound drainage, separation, fever, and WBC count. If the wound is infected, the wound should be opened, explored, drained, irrigated, debridement, and dressed open then; when the infection has cleared and there is granulation tissue growth, the wound can be closed by secondary intervention [8]. Antibiotic therapy is only used for wound infections associated with cellulitis or edema. Superficial incisional infections that have been opened can usually be managed without antibiotics if there is no associated cellulitis [5].

When closing a wound, if there is a large incision, staples are usually used because they are less reactive than sutures and have a better end result; however, sutures are utilized when there might be tension on the skin to distribute it easily. Staples are also less likely to obscure wound drainage and impending separation than subcuticular sutures, and if part of the wound is infected with staples, only remove the selected staples without opening the entire skin incisions as it generally happens once a subcuticular stitch is cut [4]. The distance between the placements of staples is what determines how the wound will drain. The wound should stop draining in about 2–3 days; when it does, it should be left uncovered. If it continues to drain, it can be kept covered and the dressings changed daily until it stops draining.

Complications from surgery like bleeding beyond a certain period say 10 days from the incision site does not really happen after laparoscopic surgery. Any bleeding usually happens early; the trocar can tamponade during the surgery and can injure a vessel. The bleeding depends on where the incision site is. The subcuticular superficial arteries can bleed or the inferior epigastric vessel if there was a lateral incision made. Usually the nurse will inform the doctor that the dressings are saturated and a pressure dressing is placed until the bleeding stops.

Some other complications from surgery include seromas, hematomas, fascial dehiscence, hernias, evisceration, and nerve injury. Seromas and hematomas are not common with laparoscopic incisions and seen more with open surgery; when a seroma or hematoma has occurred, it can be aspirated, and if a seroma recurs, it should be aspirated until it's fully gone. The wound can also have dehiscence; the main reasons for wound dehiscence are failure of the suture to remain anchored to the fascia, suture damage, knot failure, and stiches applied too close together. In 95% of abdominal wall dehiscence, the sutures and knots are intact, but the suture has pulled through the necrotic fascia due to the sutures being too close to the edge of a wound or under too much tension. If fascial disruption is suspected, wound exploration should be done in the operating room; complete dehiscence is a surgical emergency and is associated with a 10% mortality rate [4]. Management of SSI depends on the severity of the infection. Minor and superficial infections may be treated with antibiotic therapy. If the infection is deep and severe with pus or fluid drainage, the wound should be opened, explored, drained, irrigated, debrided, and dressed open, and the dressing should be changed daily or more often if the drainage is severe. When the infection has cleared and there is granulation tissue growth, the wound can be closed by secondary intention [8]. Antibiotic therapy is only used for wound infections associated with cellulitis or edema. Superficial incisional infections that have been opened can usually be managed without antibiotics if there is no associated cellulitis [5].

### 6. Wound dehiscence

5. Postoperative wound infections

210 Wound Healing - Current Perspectives

Surgical site infections are defined as infections that occur 30 days after surgery with no implant or within 1 year if an implant is placed and infection appears to be related to surgery. Surgical site infections (SSI) are seen in about 4% of clean wounds and 35% of contaminated wounds, so they are generally rare. However, certain risk factors predispose a patient to an SSI, which include diabetes, obesity, immunosuppression, cardiovascular disease, smoking, cancer, preventative surgery, malnutrition, and prior irradiation [8]. Obese patients are at increased risk especially if the incision is in the umbilical area due to fatty tissue not being well vascularized, the difficulty of cleaning it, and usually multiple incisions being made in obese patients. SSIs are associated with substantial morbidity and mortality. Patients with SSI are twice as likely to die, 60% more likely to be admitted to the intensive care unit, and more than

Signs and symptoms of incisional infection vary significantly depending on the types, severity, and pathogens. The most frequent symptoms include fever, feeling of malaise, fluid drainage, increased wound pain, redness and swelling around the wound, and loss of function and movement. SSI may be caused either by endogenous or exogenous microorganisms. Most SSIs are caused by endogenous microorganisms present on the patient's skin at the site of surgical incision, and Gram-positive bacteria such as Staphylococcus aureus are the most common microorganisms. SSI may also be caused by organisms within the patient's body that are exposed during surgery, such as Gram-negative microorganisms in the gastrointestinal tract. Exogenous sources of microorganisms include surgical instruments, operating room surfaces, the air, and personnel. Usually all wound infections happen on day 5 after surgery, and it's safe to suspect a staph infection due to the commonality of it. Of the rarer types of infections, Group A strep is seen on day 2 and clostridium usually is seen on day 3. To diagnose surgical site infections, a clinician should look for redness, induration, warmth, pain, purulent wound drainage, separation, fever, and WBC count. If the wound is infected, the wound should be opened, explored, drained, irrigated, debridement, and dressed open then; when the infection has cleared and there is granulation tissue growth, the wound can be closed by secondary intervention [8]. Antibiotic therapy is only used for wound infections associated with cellulitis or edema. Superficial incisional infections that have been opened can usually be managed without antibiotics if there is no associated cellulitis [5]. When closing a wound, if there is a large incision, staples are usually used because they are less reactive than sutures and have a better end result; however, sutures are utilized when there might be tension on the skin to distribute it easily. Staples are also less likely to obscure wound drainage and impending separation than subcuticular sutures, and if part of the wound is infected with staples, only remove the selected staples without opening the entire skin incisions as it generally happens once a subcuticular stitch is cut [4]. The distance between the placements of staples is what determines how the wound will drain. The wound should stop draining in about 2–3 days; when it does, it should be left uncovered. If it continues to drain, it

five times more likely to be readmitted to the hospital after discharge.

can be kept covered and the dressings changed daily until it stops draining.

Complications from surgery like bleeding beyond a certain period say 10 days from the incision site does not really happen after laparoscopic surgery. Any bleeding usually happens early; the trocar can tamponade during the surgery and can injure a vessel. The bleeding The quality of postoperative wound healing plays a significant role and is critical in patient recovery and rehabilitation because mortality and morbidity rates are affected by surgical wound dehiscence (SWD). SWD is defined as partial or total disruption of any or all layers of operative wounds—from simple skin dehiscence and hernia formation to the most severe and potentially lethal forms characterized by evisceration, gastrointestinal anastomotic leaks, pancreatic fistulas, and vascular pseudoaneurysms. The impact of SWD can be considerable: increased mortality, delayed hospital discharge, readmission, future surgery, delayed adjuvant treatment, suboptimal esthetic outcome, and impaired psychosocial well-being. SWD occurrence rates can vary significantly, from 0.65 to 2.1% in sternotomy to as high as 16.9–41.8% in pilonidal sinus surgery, and the cost for these wounds was \$13.1 billion according to Medicare data from a 2014 report.

### 7. Bleeding and hematoma

Bleeding is a relatively rare complication if the wound is inspected carefully before closing. Bleeding usually happens early; the trocar can tamponade during the surgery and can injure a vessel. When the blood comes out from the wound, it can be seen. However, if the blood is inside the wound, it causes hematoma or seroma. Management depends on the site and severity of the bleeding. The hematoma should be decompressed and blood is removed. If a relatively larger vessel is involved and still shows active bleeding, a suture should be used to stop it, but this is rare. For minor bleeding, a pressure dressing is placed until the bleeding stops.

References

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[2] Zhao Y, Paderu P, Delmas G, Dolgov E, Lee MH, Senter M, et al. Carbohydrate-derived fulvic acid is a highly promising topical agent to enhance healing of wounds infected with drug-resistant pathogens. Journal of Trauma and Acute Care Surgery. 2015;79(4 Suppl 2):

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[4] Macintyre IM, Wilson RG. Laparoscopic cholecystectomy. The British Journal of Surgery.

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[6] Applied Medical Gel Point Access Platforms. Applied Medical Resources Corporation. 2016. Available from: http://www.appliedmedical.com/Products/GelPoint\_Overview.aspx

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[9] Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes.

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midline incisions. Southern Medical Journal. 1995;88(4):450-453

### 8. Concluding remarks

Risk factors for most young and healthy patients and surgical wound complications are rare when the surgery is done carefully. However, risk factors for developing wound complications exist in various patients, and surgical procedure itself can also play a major role in developing complications. This includes older age, diabetes, renal diseases, the use of tobacco products and steroids, compromised immune system, obesity, poor nutritional status, and bacterial infection or colonization at a remote body sites [9–12]. To reduce wound complications, the surgical team plays a critical role. This includes adequate preoperative preparation to improve patient's overall health, careful surgical site selection, accurate procedure with as little collateral damage as possible, careful wound closure, and postoperative management. A high-quality multidisciplinary team should be able to perform safe surgeries with little complications even in some health-compromised patients.

### Acknowledgements

The authors thank Dr. Andrei Stieber, general surgeon at DeKalb Medical, for sharing his insights from his surgical practice in wound care.

### Conflict of interests to declare

None.

### Author details

Rebekah Amarini<sup>1</sup> , Sufan Chien2,3 and Girish J. Kotwal<sup>2</sup> \*

\*Address all correspondence to: gjkotw01@gmail.com

1 Hoboken University Medical Center, Carepoint Health System, Hoboken, NJ, USA

2 Noveratech Louisville, KY, USA

3 Department of Surgery, Price Institute of Surgical Research, University of Louisville School of Medicine, Louisville, KY, USA

## References

vessel. When the blood comes out from the wound, it can be seen. However, if the blood is inside the wound, it causes hematoma or seroma. Management depends on the site and severity of the bleeding. The hematoma should be decompressed and blood is removed. If a relatively larger vessel is involved and still shows active bleeding, a suture should be used to stop it, but this is

Risk factors for most young and healthy patients and surgical wound complications are rare when the surgery is done carefully. However, risk factors for developing wound complications exist in various patients, and surgical procedure itself can also play a major role in developing complications. This includes older age, diabetes, renal diseases, the use of tobacco products and steroids, compromised immune system, obesity, poor nutritional status, and bacterial infection or colonization at a remote body sites [9–12]. To reduce wound complications, the surgical team plays a critical role. This includes adequate preoperative preparation to improve patient's overall health, careful surgical site selection, accurate procedure with as little collateral damage as possible, careful wound closure, and postoperative management. A high-quality multidisciplinary team should be able to perform safe surgeries with little complications even

The authors thank Dr. Andrei Stieber, general surgeon at DeKalb Medical, for sharing his

\*

, Sufan Chien2,3 and Girish J. Kotwal<sup>2</sup>

1 Hoboken University Medical Center, Carepoint Health System, Hoboken, NJ, USA

3 Department of Surgery, Price Institute of Surgical Research, University of Louisville School

rare. For minor bleeding, a pressure dressing is placed until the bleeding stops.

8. Concluding remarks

212 Wound Healing - Current Perspectives

in some health-compromised patients.

Conflict of interests to declare

2 Noveratech Louisville, KY, USA

of Medicine, Louisville, KY, USA

insights from his surgical practice in wound care.

\*Address all correspondence to: gjkotw01@gmail.com

Acknowledgements

None.

Author details

Rebekah Amarini<sup>1</sup>


**Chapter 13**

**Provisional chapter**

**Multidisciplinary Approaches to the Stimulation of**

**Multidisciplinary Approaches to the Stimulation of** 

**Research focus:** Skin injuries are evolving as an epidemic issue. Chronic skin lesion is a globally widespread disease, often referred to as a "wound difficult to heal" and one which has a strong impact on both overall health and quality of life. Genetic and clinical variables, such as diabetes, smoking and inflammatory/immunological pathologies, are among the important risk factors limiting the regenerative powers of many therapeutic applications. Therefore, optimisation of current clinical strategies is critical. **Experimental research:** Here we summarise the field's current state by focusing on the use of stem-cell therapeutic applications in wound healing, placing considerable emphasis on current clinical approaches being developed at Rome's *Sapienza* University. These involve protocols for the ex vivo expansion of adipose tissue-derived mesenchyme stem cells using a patented GMP-compliant platelet lysate, Mesengen™, and cellular and acellular dermal substitutes. A combination of multiple strategies, including genetic modifications of stem cells, biomimetic scaffolds or novel vehicles like nanoparticles, is also discussed as future approaches. **Case studies:** Here we present a report portraying our clinical experience of the treatment of chronic phlebostatic ulcers. The aim of the study reported here was to evaluate the effectiveness of treatment with dermal substitutes of cutaneous lesions originating from chronic venous insufficiency, therapy which took into consideration parameters such as the reduction of wound size and the improvement of quality of life. Chronic skin lesion, a globally widespread disease, is often referred to as a "difficult wound" and has a strong impact on both overall health and quality of life. The difficulties encountered when seeking to heal this ailment have led to a quest for and development of new therapeutic approaches, including dermal substitutes. We can subdivide these into acellular matrices, such as Integra and Hyalomatrix, and cell therapies such as platelet concentrate and mesenchyme cell concentrate. **Results:** In all the patients treated, elements of improvement

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.81791

**Wound Healing and Use of Dermal Substitutes in**

**Wound Healing and Use of Dermal Substitutes in** 

**Chronic Phlebostatic Ulcers**

**Chronic Phlebostatic Ulcers**

Bruno Salvati

Bruno Salvati

**Abstract**

Raffaele Capoano, Rita Businaro, Besar Kolce, Andrea Biancucci, Silvia Izzo, Lidia De Felice and

Raffaele Capoano, Rita Businaro, Besar Kolce, Andrea Biancucci, Silvia Izzo, Lidia De Felice and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81791

#### **Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal Substitutes in Chronic Phlebostatic Ulcers Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal Substitutes in Chronic Phlebostatic Ulcers**

DOI: 10.5772/intechopen.81791

Raffaele Capoano, Rita Businaro, Besar Kolce, Andrea Biancucci, Silvia Izzo, Lidia De Felice and Bruno Salvati Raffaele Capoano, Rita Businaro, Besar Kolce, Andrea Biancucci, Silvia Izzo, Lidia De Felice and Bruno Salvati

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.81791

#### **Abstract**

**Research focus:** Skin injuries are evolving as an epidemic issue. Chronic skin lesion is a globally widespread disease, often referred to as a "wound difficult to heal" and one which has a strong impact on both overall health and quality of life. Genetic and clinical variables, such as diabetes, smoking and inflammatory/immunological pathologies, are among the important risk factors limiting the regenerative powers of many therapeutic applications. Therefore, optimisation of current clinical strategies is critical. **Experimental research:** Here we summarise the field's current state by focusing on the use of stem-cell therapeutic applications in wound healing, placing considerable emphasis on current clinical approaches being developed at Rome's *Sapienza* University. These involve protocols for the ex vivo expansion of adipose tissue-derived mesenchyme stem cells using a patented GMP-compliant platelet lysate, Mesengen™, and cellular and acellular dermal substitutes. A combination of multiple strategies, including genetic modifications of stem cells, biomimetic scaffolds or novel vehicles like nanoparticles, is also discussed as future approaches. **Case studies:** Here we present a report portraying our clinical experience of the treatment of chronic phlebostatic ulcers. The aim of the study reported here was to evaluate the effectiveness of treatment with dermal substitutes of cutaneous lesions originating from chronic venous insufficiency, therapy which took into consideration parameters such as the reduction of wound size and the improvement of quality of life. Chronic skin lesion, a globally widespread disease, is often referred to as a "difficult wound" and has a strong impact on both overall health and quality of life. The difficulties encountered when seeking to heal this ailment have led to a quest for and development of new therapeutic approaches, including dermal substitutes. We can subdivide these into acellular matrices, such as Integra and Hyalomatrix, and cell therapies such as platelet concentrate and mesenchyme cell concentrate. **Results:** In all the patients treated, elements of improvement

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

were observed: the appearance on the wound bed of small islands of granulation tissue, superficialization of the bottom of the ulcer and a growth of marginal tissue. During the first 30 days, a reduction in more than 25% of the area of the lesion and a reduction in more than 50% at the end of the observation period were recorded in 10 of the patients who underwent preliminary surgical treatment out of the 13 subjects included in the study sample. On the whole, at the end of the observation period, we witnessed an average 57% decrease in the lesion in all the patients; furthermore, during the treatment period, there was a gradual reduction in pain, measured using the NRS numerical scale. An overall average reduction in pain of four points on the NRS numerical scale was achieved. At the end of the 8-week evaluation period, the majority of the patients reported an improvement in the quality of their lives, since, in addition to the reduction of spontaneous pain, there was a diminution of pruritus, secretions—often malodorous and capable of affecting social life negatively—with recovery of functional capacity and almost complete recovery of habitual daily activities. During the period of treatment, no superinfections of the wounds or secondary complications related to the use of the various products were detected. **Main conclusions:** The numerous technological opportunities provided by regenerative medicine—including advanced dressings and dermal substitutes—if applied correctly, in compliance with a multidisciplinary approach where necessary, seem to offer advantages not only in terms of clinical efficacy and patient life quality but also in terms, it would appear, of healthcare costs, an aspect which should not be either overlooked or underestimated.

Both clinical and genetic features of individual patients must be considered when addressing wound healing, as well as variations in medical responses based on the type of chemical tools employed and the nature and extent of the injured area. In fact, large wounds, under either adverse local or systemic conditions, respond poorly to treatment and can frequently reopen. It is calculated that about 10% of the population is likely to develop a chronic lesion during its lifetime, leading to discomfort (pain, reduced hygiene, sleep disturbance), loss of autonomy, need of assistance and frequent hospitalisation, with considerable deterioration of the quality of life (QoL) due, among other things, to embarrassment, social isolation, compromisation of employability, monetary costs, anxiety-depressive syndromes [5] as well as a mortality rate of 2.5% [6, 7]. A chronic dermal lesion means having to live with a persistent, enduring and treatment-resistant wound, which has a significant impact on the overall health and quality

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

http://dx.doi.org/10.5772/intechopen.81791

217

Chronic venous insufficiency is responsible for 80% of the ulcers affecting the lower limbs, a

As people grow older, venous ulcers begin to constitute a serious health problem, considering the fact that 4% of people aged over 65 suffer from them. This renders the whole issue highly topical while making possible therapeutic treatments within the ambit of regenerative

A number of strategies have been developed recently to treat dermal wounds resulting from chemical exposure. One of the most efficient methods used to reduce bacterial load and the incidence of sepsis is debridement of the wound [8]. Cleansing agents and topical antibiotics

Current literature contains numerous studies [10–12], which underline the diffusion of medication considered "advanced" and capable of producing improved clinical and economic outcomes associated with the healing of similar lesions. The notion "advanced" implies not only the use of particular products and medications, surpassing the therapeutic concept of keeping the ulcer in a "dry" environment and leaving medication on the lesion for lengthy periods of time, but also means changing wound management substantially. Advanced methodologies seem to lead to a diminution of the number of medication sessions required compared to those prescribed by traditional approaches and, theoretically speaking, a reduction in overall healing time, which also spells a reduction in costs. We have expressed ourselves in dubitative terms here because many factors of a logistic and not simply clinical nature, capable of impacting negatively or positively on the treatment opted for, are involved and can contribute to the success or failure of the therapy. One of the factors—common to acute and chronic lesions and, therefore, regardless of aetiopathogenesis—which is capable of having a positive impact on regenerative therapy is appropriate "wound bed preparation" (WBP). "Wound bed preparation" involves a detailed, coordinated and sometimes multidisciplinary management of the lesion aimed at removing all the factors, which may hamper tissue regeneration, while favouring endogenous healing, promoting cellular proliferation and the reparatory processes "triggered" by the products applied. This concept, no longer considered recent, has significantly affected the management of chronic cutaneous wounds and the results obtained.

are also useful when seeking to reduce microbial growth and invasive infection [9].

sequela constituting one of the gravest complications encountered by CVIs [2].

of life of patients, members of their families and caregivers.

medicine of considerable interest.

**Keywords:** wound healing, skin lesion, regenerative medicine, dermal substitutes, phlebostatic ulcers

### **1. Introduction**

The management of scarring in chronic wounds represents one of the most relevant clinical problems affecting healthcare in the United States and in Europe [1]. Scars can cause severe physical damage, resulting, predominantly, in damage to the skin.

This kind of lesion causes loss of cutaneous substance, varies in size and involves loss of underlying tissue as well; this kind of lesion is often defined as "difficult" because it fails to heal spontaneously or achieve speedy and complete recovery. Acute or chronic wounds of this type are due to multifactor pathogeneses, and their healing is impeded, as a rule, by persistent local or systemic factors which favour chronicisation.

Medical-surgical treatment of these so-called "difficult" lesions represents a constantly increasing social-health issue, a "silent epidemic" affecting large sectors of the world's population, one which, at present, concerns about 2,000,000 Italians; these numbers are destined to grow further due not only to diverse risk factors but also to the phenomenon of ageing [2, 3]. Subjects with pre-existing diseases are of particular concern, as treatment and resolution of injury frequently require long-term care. Healing is often compromised in similar individuals because of the presence of diabetes, the metabolic syndrome, chronic renal failure and ageing [4] since the ability to rapidly re-epithelialise and revascularise injured tissue is impaired. In some cases, the evolution of the lesion is correlated to the root-cause pathology, as, in particular, in the case of "immunohaematological" ulcers.

Both clinical and genetic features of individual patients must be considered when addressing wound healing, as well as variations in medical responses based on the type of chemical tools employed and the nature and extent of the injured area. In fact, large wounds, under either adverse local or systemic conditions, respond poorly to treatment and can frequently reopen.

were observed: the appearance on the wound bed of small islands of granulation tissue, superficialization of the bottom of the ulcer and a growth of marginal tissue. During the first 30 days, a reduction in more than 25% of the area of the lesion and a reduction in more than 50% at the end of the observation period were recorded in 10 of the patients who underwent preliminary surgical treatment out of the 13 subjects included in the study sample. On the whole, at the end of the observation period, we witnessed an average 57% decrease in the lesion in all the patients; furthermore, during the treatment period, there was a gradual reduction in pain, measured using the NRS numerical scale. An overall average reduction in pain of four points on the NRS numerical scale was achieved. At the end of the 8-week evaluation period, the majority of the patients reported an improvement in the quality of their lives, since, in addition to the reduction of spontaneous pain, there was a diminution of pruritus, secretions—often malodorous and capable of affecting social life negatively—with recovery of functional capacity and almost complete recovery of habitual daily activities. During the period of treatment, no superinfections of the wounds or secondary complications related to the use of the various products were detected. **Main conclusions:** The numerous technological opportunities provided by regenerative medicine—including advanced dressings and dermal substitutes—if applied correctly, in compliance with a multidisciplinary approach where necessary, seem to offer advantages not only in terms of clinical efficacy and patient life quality but also in terms, it would appear, of healthcare costs, an aspect which should not be either overlooked or underestimated.

**Keywords:** wound healing, skin lesion, regenerative medicine, dermal substitutes,

The management of scarring in chronic wounds represents one of the most relevant clinical problems affecting healthcare in the United States and in Europe [1]. Scars can cause severe

This kind of lesion causes loss of cutaneous substance, varies in size and involves loss of underlying tissue as well; this kind of lesion is often defined as "difficult" because it fails to heal spontaneously or achieve speedy and complete recovery. Acute or chronic wounds of this type are due to multifactor pathogeneses, and their healing is impeded, as a rule, by

Medical-surgical treatment of these so-called "difficult" lesions represents a constantly increasing social-health issue, a "silent epidemic" affecting large sectors of the world's population, one which, at present, concerns about 2,000,000 Italians; these numbers are destined to grow further due not only to diverse risk factors but also to the phenomenon of ageing [2, 3]. Subjects with pre-existing diseases are of particular concern, as treatment and resolution of injury frequently require long-term care. Healing is often compromised in similar individuals because of the presence of diabetes, the metabolic syndrome, chronic renal failure and ageing [4] since the ability to rapidly re-epithelialise and revascularise injured tissue is impaired. In some cases, the evolution of the lesion is correlated to the root-cause pathology, as, in particu-

physical damage, resulting, predominantly, in damage to the skin.

persistent local or systemic factors which favour chronicisation.

lar, in the case of "immunohaematological" ulcers.

phlebostatic ulcers

216 Wound Healing - Current Perspectives

**1. Introduction**

It is calculated that about 10% of the population is likely to develop a chronic lesion during its lifetime, leading to discomfort (pain, reduced hygiene, sleep disturbance), loss of autonomy, need of assistance and frequent hospitalisation, with considerable deterioration of the quality of life (QoL) due, among other things, to embarrassment, social isolation, compromisation of employability, monetary costs, anxiety-depressive syndromes [5] as well as a mortality rate of 2.5% [6, 7]. A chronic dermal lesion means having to live with a persistent, enduring and treatment-resistant wound, which has a significant impact on the overall health and quality of life of patients, members of their families and caregivers.

Chronic venous insufficiency is responsible for 80% of the ulcers affecting the lower limbs, a sequela constituting one of the gravest complications encountered by CVIs [2].

As people grow older, venous ulcers begin to constitute a serious health problem, considering the fact that 4% of people aged over 65 suffer from them. This renders the whole issue highly topical while making possible therapeutic treatments within the ambit of regenerative medicine of considerable interest.

A number of strategies have been developed recently to treat dermal wounds resulting from chemical exposure. One of the most efficient methods used to reduce bacterial load and the incidence of sepsis is debridement of the wound [8]. Cleansing agents and topical antibiotics are also useful when seeking to reduce microbial growth and invasive infection [9].

Current literature contains numerous studies [10–12], which underline the diffusion of medication considered "advanced" and capable of producing improved clinical and economic outcomes associated with the healing of similar lesions. The notion "advanced" implies not only the use of particular products and medications, surpassing the therapeutic concept of keeping the ulcer in a "dry" environment and leaving medication on the lesion for lengthy periods of time, but also means changing wound management substantially. Advanced methodologies seem to lead to a diminution of the number of medication sessions required compared to those prescribed by traditional approaches and, theoretically speaking, a reduction in overall healing time, which also spells a reduction in costs. We have expressed ourselves in dubitative terms here because many factors of a logistic and not simply clinical nature, capable of impacting negatively or positively on the treatment opted for, are involved and can contribute to the success or failure of the therapy. One of the factors—common to acute and chronic lesions and, therefore, regardless of aetiopathogenesis—which is capable of having a positive impact on regenerative therapy is appropriate "wound bed preparation" (WBP). "Wound bed preparation" involves a detailed, coordinated and sometimes multidisciplinary management of the lesion aimed at removing all the factors, which may hamper tissue regeneration, while favouring endogenous healing, promoting cellular proliferation and the reparatory processes "triggered" by the products applied. This concept, no longer considered recent, has significantly affected the management of chronic cutaneous wounds and the results obtained.

In order to outline the principles of WBP more precisely, the English acronym TIME (tissue, infection or inflammation, moisture imbalance, epidermal margin) is used. TIME breaks up what is actually a single therapeutic process into parts, indicating the fundamental aspects to be dealt with during preparation of the wound bed.

factors associated with cell survival, proliferation and differentiation [19]. They can consist in synthetic or absorbable polymeric materials present in nature which may be biological,

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

http://dx.doi.org/10.5772/intechopen.81791

219

The four main approaches to scaffolding include the use of ECM-secreting cell sheets; preconstituted porous scaffolds of synthetic, natural and biodegradable biomaterials; decellularised

In the field of surface-tissue regeneration, numerous grafts based on acellular dermal and epidermal scaffolds have been tested, using natural and synthetic polymers, or a combination

Another important area where regenerative medicine is applied is that of cell therapy, which is based on the autologous cell suspension (SCAut) technique, that is, the exploitation of cells

A new chapter in the history of regenerative medicine, though still very controversial and limited to specifically clinical application, is that of tissue bioengineering, that is, the use of totipotent, pluripotent and unipotent cells, potentially capable of originating, respectively, any kind of tissue, a wide range of tissues and a sole cell line; the cells chosen for cropping can come from autologous, homologous and heterologous samples. The isolation, culture and maturation of stem cells are recurred to with a view to replacing damaged tissue. Among the first mechanical and chemical processing techniques used is the in vitro cultivation of the epidermis using the "feeder-layer" methodology proposed by Rheinwald J.G. and Green H. in 1977 and the grafting of laminates of expanded in vitro keratinocytes or keratinocyte suspensions onto de-epidermised human dermis (DED) introduced by Cuono C.B. in 1987. The potential use of different types of stem cells for regenerative skin-lesion repair has recently received considerable attention [21]. "Difficult" or "stubborn" chronic wounds, characterised by extensive loss of substance and an enduring clinical history of healing and recurrence, represent one of the fields where the bioengineering of tissue may be applied and one of the discipline's main areas

The cell lines which arouse the greatest interest at the moment are those taken from the embryo, the foetus and the umbilical cord as well as a number of adult cells like adipose-

Several protocols have been established aimed at ensuring the resolution of wound issues by targeting different phases of the healing process, namely, control of inflammation in a suitable microenvironment, enhancement of stem-cell engraftment after implantation, efficient and terminal transdifferentiation of progenitors towards dermal lineages and the reconstruction of the vasculature system surrounding the wound [22, 23]. Mesenchymal stem cells (MSCs) have recently been proposed as a promising solution capable of enhancing the re-epithelialisation phase [24]. Studies using mouse models have shown that the intradermal injection of human MSCs or adipose-tissue-derived stromal cells (ASCs) accelerates skin-wound healing in nude mice [1]. Similarly, results of clinical trials have demonstrated the benefits derived from the employment of both autologous or heterologous MSCs, especially in chronic wounds [25–28]. Defined as adult multipotent cells, MSCs can be easily obtained from multiple sources, including

degradable or nondegradable.

taken from the patients themselves.

tissue-derived mesenchymal stem cells.

of challenge.

ECM scaffolds and cells enveloped in hydrogel [20].

of both, described as "effective substitutes for wound healing".

In lesion management, WBP permits definitive elimination of all the elements that may hinder the development of granulation tissue, thus laying the foundations for effective use of innovative therapeutic tools. The purpose of advanced dressing is to create an environment ideal for proliferative cicatrisation processes, for the isolation of the wound from traumatic and infectious agents, while improving the state of the bottom of the wound and promoting maximum possible tropism of the margins and the periwound skin.

Additionally, treatment of the wound with autologous leukocytes seeded in a proangiogenic matrix and enriched with a platelet concentrate preparation has been reported to induce the release of growth factors, cytokines and chemokines, thus increasing the in situ recruitment of endothelial precursor cells and promoting the resolution of microbial infections [13]. Despite these improvements, treatment of dermal wounds has not always produced positive outcomes. Major drawbacks include the fact that the skin is a highly complex organ and is, therefore, difficult to reconstruct after injury. In actual fact, the physiological re-epithelialisation phase is a multistep process involving several cell types and molecular mechanisms, and the presence of a favourable environment for bacterial colonisation is highly undesirable [14, 15]. As a consequence, most current treatments have been palliative only, aiming mainly at accelerating healing time and limiting additional clinical complications caused by fortuitous bacterial infection. Therefore, alternative strategies are required to balance the treatment of patients, economic costs and the safety of civilians.

The main aim of regenerative medicine is to repair organs and tissues that have been damaged by pathological events and/or trauma and/or ageing in order to restore or improve their biological functions. It is a multidisciplinary field undergoing rapid growth and involving the medical, humanistic and engineering sciences, a field which endeavours to develop functional cells and substitute tissues or organs with a view to repairing, replacing or improving the biological functions lost due to congenital anomalies, trauma and illness or as a result of ageing. The increase in average life expectancy has led, actually, to the need to protract the time people spend in active employment and has made the physical and mental efficiency of older subjects mandatory, but it also implies an inevitable rise in incidences of neoplasms and pathologies that cannot always be cured by the therapies available at present. From this stems the need to develop therapies capable of replacing or regenerating organs damaged by pathological processes or traumas. Referring specifically to the present text, regenerative medicine can solve the problem of chronic vascular ulcers. Some lesions can benefit particularly from regenerative, cellulated and acellulated materials as well as biochemical supports (scaffolds) used to "trigger" granulation, repair tissue and, therefore, cover wounds.

Scaffolds, according to the definition of tissue engineering provided by the National Science, are materials that can best attend to the restoration, maintenance and improvement of the function of tissues, playing a unique role in their repair and above all, in their regeneration [16–18]. Scaffolds provide an appropriate platform for the essential provision of various factors associated with cell survival, proliferation and differentiation [19]. They can consist in synthetic or absorbable polymeric materials present in nature which may be biological, degradable or nondegradable.

In order to outline the principles of WBP more precisely, the English acronym TIME (tissue, infection or inflammation, moisture imbalance, epidermal margin) is used. TIME breaks up what is actually a single therapeutic process into parts, indicating the fundamental aspects to

In lesion management, WBP permits definitive elimination of all the elements that may hinder the development of granulation tissue, thus laying the foundations for effective use of innovative therapeutic tools. The purpose of advanced dressing is to create an environment ideal for proliferative cicatrisation processes, for the isolation of the wound from traumatic and infectious agents, while improving the state of the bottom of the wound and promoting maximum

Additionally, treatment of the wound with autologous leukocytes seeded in a proangiogenic matrix and enriched with a platelet concentrate preparation has been reported to induce the release of growth factors, cytokines and chemokines, thus increasing the in situ recruitment of endothelial precursor cells and promoting the resolution of microbial infections [13]. Despite these improvements, treatment of dermal wounds has not always produced positive outcomes. Major drawbacks include the fact that the skin is a highly complex organ and is, therefore, difficult to reconstruct after injury. In actual fact, the physiological re-epithelialisation phase is a multistep process involving several cell types and molecular mechanisms, and the presence of a favourable environment for bacterial colonisation is highly undesirable [14, 15]. As a consequence, most current treatments have been palliative only, aiming mainly at accelerating healing time and limiting additional clinical complications caused by fortuitous bacterial infection. Therefore, alternative strategies are required

to balance the treatment of patients, economic costs and the safety of civilians.

used to "trigger" granulation, repair tissue and, therefore, cover wounds.

The main aim of regenerative medicine is to repair organs and tissues that have been damaged by pathological events and/or trauma and/or ageing in order to restore or improve their biological functions. It is a multidisciplinary field undergoing rapid growth and involving the medical, humanistic and engineering sciences, a field which endeavours to develop functional cells and substitute tissues or organs with a view to repairing, replacing or improving the biological functions lost due to congenital anomalies, trauma and illness or as a result of ageing. The increase in average life expectancy has led, actually, to the need to protract the time people spend in active employment and has made the physical and mental efficiency of older subjects mandatory, but it also implies an inevitable rise in incidences of neoplasms and pathologies that cannot always be cured by the therapies available at present. From this stems the need to develop therapies capable of replacing or regenerating organs damaged by pathological processes or traumas. Referring specifically to the present text, regenerative medicine can solve the problem of chronic vascular ulcers. Some lesions can benefit particularly from regenerative, cellulated and acellulated materials as well as biochemical supports (scaffolds)

Scaffolds, according to the definition of tissue engineering provided by the National Science, are materials that can best attend to the restoration, maintenance and improvement of the function of tissues, playing a unique role in their repair and above all, in their regeneration [16–18]. Scaffolds provide an appropriate platform for the essential provision of various

be dealt with during preparation of the wound bed.

218 Wound Healing - Current Perspectives

possible tropism of the margins and the periwound skin.

The four main approaches to scaffolding include the use of ECM-secreting cell sheets; preconstituted porous scaffolds of synthetic, natural and biodegradable biomaterials; decellularised ECM scaffolds and cells enveloped in hydrogel [20].

In the field of surface-tissue regeneration, numerous grafts based on acellular dermal and epidermal scaffolds have been tested, using natural and synthetic polymers, or a combination of both, described as "effective substitutes for wound healing".

Another important area where regenerative medicine is applied is that of cell therapy, which is based on the autologous cell suspension (SCAut) technique, that is, the exploitation of cells taken from the patients themselves.

A new chapter in the history of regenerative medicine, though still very controversial and limited to specifically clinical application, is that of tissue bioengineering, that is, the use of totipotent, pluripotent and unipotent cells, potentially capable of originating, respectively, any kind of tissue, a wide range of tissues and a sole cell line; the cells chosen for cropping can come from autologous, homologous and heterologous samples. The isolation, culture and maturation of stem cells are recurred to with a view to replacing damaged tissue. Among the first mechanical and chemical processing techniques used is the in vitro cultivation of the epidermis using the "feeder-layer" methodology proposed by Rheinwald J.G. and Green H. in 1977 and the grafting of laminates of expanded in vitro keratinocytes or keratinocyte suspensions onto de-epidermised human dermis (DED) introduced by Cuono C.B. in 1987. The potential use of different types of stem cells for regenerative skin-lesion repair has recently received considerable attention [21]. "Difficult" or "stubborn" chronic wounds, characterised by extensive loss of substance and an enduring clinical history of healing and recurrence, represent one of the fields where the bioengineering of tissue may be applied and one of the discipline's main areas of challenge.

The cell lines which arouse the greatest interest at the moment are those taken from the embryo, the foetus and the umbilical cord as well as a number of adult cells like adiposetissue-derived mesenchymal stem cells.

Several protocols have been established aimed at ensuring the resolution of wound issues by targeting different phases of the healing process, namely, control of inflammation in a suitable microenvironment, enhancement of stem-cell engraftment after implantation, efficient and terminal transdifferentiation of progenitors towards dermal lineages and the reconstruction of the vasculature system surrounding the wound [22, 23]. Mesenchymal stem cells (MSCs) have recently been proposed as a promising solution capable of enhancing the re-epithelialisation phase [24]. Studies using mouse models have shown that the intradermal injection of human MSCs or adipose-tissue-derived stromal cells (ASCs) accelerates skin-wound healing in nude mice [1]. Similarly, results of clinical trials have demonstrated the benefits derived from the employment of both autologous or heterologous MSCs, especially in chronic wounds [25–28]. Defined as adult multipotent cells, MSCs can be easily obtained from multiple sources, including adipose tissue deposits localised in different areas of the body and gathered during major and/ or aesthetic surgical procedures [29, 30]. Multiple mechanisms underlying the potential ability of both populations to influence wound repair positively have been proposed; these include modulation of inflammatory states, stimulation of angiogenesis, cell proliferation and fibroblast activity, activation and enhanced migration of keratinocytes to sites of injury in a paracrine fashion, possible direct transdifferentiation of MSCs towards dermal lineage (including fibroblasts and keratinocytes) and, finally, the recruitment of host cells [25, 31, 32]. After in vivo administration, the immune tolerance generated by ASCs, defined as the ability to modulate the immune-surveillance system in the recipient, has been largely reported as their chief biological property, thus highlighting one important advantage their use brings [33, 34]. Moreover, cross communication between ASCs and inflammatory cells at the site of an injury is a major contributory factor. Soluble factors released by MSCs and ASCs, such as vascular endothelial growth factor, interleukin-6 or transforming growth factors, are known to regulate local cellular responses during cutaneous injury [24]. It has been noted that MSCs may also exert antibacterial effects at the site of a wound both by secreting an antimicrobial protein, IL-37, directly and by influencing immune-system phagocytosis positively [24, 35]. The proliferative and transdifferentiative potential of MSCs has been highlighted also in tissue-engineering-based applications, specifically with regard to skin graft reconstruction, where MSCs are employed either alone, as a feeder layer for keratinocytes or seeded in combination with gelatine-, collagen-/ chitosan- or fibrin polymer-based scaffolds [36–38]. Of note among suitable substrates, synthetic polymers have been shown to possess considerable ability to absorb and transport fluids as well as provide protection against bacterial exposure [39]. Other methods used to deliver MSCs to the wound site include injection and local or systemic administration of a range of conveyers such as scaffolds, matrices and human amniotic membrane grafts [40–43].

Recently, a GMP-compliant PL (Mesengen™, Pub. No. WO/2013/042095) has been developed as an adjuvant for culturing human ASCs, endothelial progenitor cells and fibroblasts [29, 46,

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The Mesengen™ generation method has been standardised and optimised so as to determine the amounts of cytokines and growth factors in the preparation. Importantly, potential fungi, viruses and bacteria known to contaminate human haemoderivatives are avoided by rapidly inactivating the Mesengen™ by means of a combination of a photochemical agent and UV radiation. A summary of the basic steps in the preparation of PL is provided in **Figure 1**. It is worth noting that researchers at *Sapienza* have exploited the biological and molecular properties of Mesengen™ by concurrently establishing a standardised protocol (**Figure 2**) to isolate and expand ex vivo ASCs from alternative fat deposits like the mediastinum (**Figure 3**) [29, 47]. Recent studies on Mesengen™ carried out by our team have also elucidated its ability to influence the commitment of ASCs by inducing epigenetic modifications [47] as well as positively altering the in vitro microenvironment by decreasing oxidative stress [46]. These studies highlight the ability of PL to boost the biological and functional properties of mesenchymal-like cell populations. Therefore, it is plausible that the combination of Mesengen™ and ASCs or other progenitor-cell populations might be employed successfully to target wound repair and regeneration. Furthermore, PL has been reported to maintain its properties either as a liquid formulation or frozen, highlighting an important clinical advantage. In the future, this approach might be considered complementary to routine strategies developed at Rome's *Sapienza* University, where a centre of excellence for

**Figure 1.** Overview of the major steps in the manufacturing of platelet lysate (Mesengen™).

51, 52].

### **2. Novel strategies developed at Rome's** *Sapienza* **University**

Despite considerable improvements in the employment of ASCs and MSCs in skin-regenerative procedures, their current use is limited because of the presence of foetal bovine serum (FBS) in the cultures during their ex vivo expansion. According to the European Union's Good Manufacturing Practice (GMP) guidelines, the employment of FBS is to be discouraged, as it is a potential source of zoonoses [29, 44, 45]. In the light of this, platelet lysate (PL), a haemoderivative enriched with soluble mitogenic factors [29, 44, 46], represents a superior alternative to FBS.Reported to enhance the biological stem-cell properties of ASCs, such as proliferation, clonogenic capacity and migration [28, 46, 47], PL has been also been recently shown to be capable of promoting ASC's pluripotency and being committed to specific phenotypes [46–49]. Interestingly, PL, manufactured in injectable form or gel [8, 9, 13, 14], embedded in scaffolds or incorporated in nanoparticles, also represents a widely investigated clinical strategy deemed to accelerate wound healing in chronic ocular and diabetic dermal ulcers. Because of the large amounts of cytokines and growth factors contained in PL, it presents multiple and significant advantages if applied locally to skin wounds when seeking enhancement of angiogenesis and fibroblast migration, restoration of collagen synthesis and reduction of oxidative stress [46]. In addition, it has been demonstrated that PL is capable of re-establishing skin integrity efficiently [50].

Recently, a GMP-compliant PL (Mesengen™, Pub. No. WO/2013/042095) has been developed as an adjuvant for culturing human ASCs, endothelial progenitor cells and fibroblasts [29, 46, 51, 52].

adipose tissue deposits localised in different areas of the body and gathered during major and/ or aesthetic surgical procedures [29, 30]. Multiple mechanisms underlying the potential ability of both populations to influence wound repair positively have been proposed; these include modulation of inflammatory states, stimulation of angiogenesis, cell proliferation and fibroblast activity, activation and enhanced migration of keratinocytes to sites of injury in a paracrine fashion, possible direct transdifferentiation of MSCs towards dermal lineage (including fibroblasts and keratinocytes) and, finally, the recruitment of host cells [25, 31, 32]. After in vivo administration, the immune tolerance generated by ASCs, defined as the ability to modulate the immune-surveillance system in the recipient, has been largely reported as their chief biological property, thus highlighting one important advantage their use brings [33, 34]. Moreover, cross communication between ASCs and inflammatory cells at the site of an injury is a major contributory factor. Soluble factors released by MSCs and ASCs, such as vascular endothelial growth factor, interleukin-6 or transforming growth factors, are known to regulate local cellular responses during cutaneous injury [24]. It has been noted that MSCs may also exert antibacterial effects at the site of a wound both by secreting an antimicrobial protein, IL-37, directly and by influencing immune-system phagocytosis positively [24, 35]. The proliferative and transdifferentiative potential of MSCs has been highlighted also in tissue-engineering-based applications, specifically with regard to skin graft reconstruction, where MSCs are employed either alone, as a feeder layer for keratinocytes or seeded in combination with gelatine-, collagen-/ chitosan- or fibrin polymer-based scaffolds [36–38]. Of note among suitable substrates, synthetic polymers have been shown to possess considerable ability to absorb and transport fluids as well as provide protection against bacterial exposure [39]. Other methods used to deliver MSCs to the wound site include injection and local or systemic administration of a range of conveyers such

220 Wound Healing - Current Perspectives

as scaffolds, matrices and human amniotic membrane grafts [40–43].

capable of re-establishing skin integrity efficiently [50].

**2. Novel strategies developed at Rome's** *Sapienza* **University**

Despite considerable improvements in the employment of ASCs and MSCs in skin-regenerative procedures, their current use is limited because of the presence of foetal bovine serum (FBS) in the cultures during their ex vivo expansion. According to the European Union's Good Manufacturing Practice (GMP) guidelines, the employment of FBS is to be discouraged, as it is a potential source of zoonoses [29, 44, 45]. In the light of this, platelet lysate (PL), a haemoderivative enriched with soluble mitogenic factors [29, 44, 46], represents a superior alternative to FBS.Reported to enhance the biological stem-cell properties of ASCs, such as proliferation, clonogenic capacity and migration [28, 46, 47], PL has been also been recently shown to be capable of promoting ASC's pluripotency and being committed to specific phenotypes [46–49]. Interestingly, PL, manufactured in injectable form or gel [8, 9, 13, 14], embedded in scaffolds or incorporated in nanoparticles, also represents a widely investigated clinical strategy deemed to accelerate wound healing in chronic ocular and diabetic dermal ulcers. Because of the large amounts of cytokines and growth factors contained in PL, it presents multiple and significant advantages if applied locally to skin wounds when seeking enhancement of angiogenesis and fibroblast migration, restoration of collagen synthesis and reduction of oxidative stress [46]. In addition, it has been demonstrated that PL is The Mesengen™ generation method has been standardised and optimised so as to determine the amounts of cytokines and growth factors in the preparation. Importantly, potential fungi, viruses and bacteria known to contaminate human haemoderivatives are avoided by rapidly inactivating the Mesengen™ by means of a combination of a photochemical agent and UV radiation. A summary of the basic steps in the preparation of PL is provided in **Figure 1**. It is worth noting that researchers at *Sapienza* have exploited the biological and molecular properties of Mesengen™ by concurrently establishing a standardised protocol (**Figure 2**) to isolate and expand ex vivo ASCs from alternative fat deposits like the mediastinum (**Figure 3**) [29, 47]. Recent studies on Mesengen™ carried out by our team have also elucidated its ability to influence the commitment of ASCs by inducing epigenetic modifications [47] as well as positively altering the in vitro microenvironment by decreasing oxidative stress [46]. These studies highlight the ability of PL to boost the biological and functional properties of mesenchymal-like cell populations. Therefore, it is plausible that the combination of Mesengen™ and ASCs or other progenitor-cell populations might be employed successfully to target wound repair and regeneration. Furthermore, PL has been reported to maintain its properties either as a liquid formulation or frozen, highlighting an important clinical advantage. In the future, this approach might be considered complementary to routine strategies developed at Rome's *Sapienza* University, where a centre of excellence for

**Figure 1.** Overview of the major steps in the manufacturing of platelet lysate (Mesengen™).

Our group's experience of advanced dressings and dermal substitutes over the years during treatment of patients with acute and chronic ulcers of multifactorial origin (arteriopathic, phlebopathic, immunological and traumatic) produced a study based on chronic arteriopathic patients, the results of which were published in the article "Wounds Difficult to Heal: An Effective Treatment Strategy" [60]. There we highlighted the fact that recognition of the aetiology of a skin lesion and the correction of the pathophysiological conditions that determine and support it are the assumption and "step" fundamental to the success of local treatment. It also emerged that a "standard", univocal treatment applicable to all and every kind of wound does not exist. Appropriate local treatment involves a combination of multiple medications, products and devices demanding respect of their timing and guarantees regarding

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

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223

In a very recent study we focused, instead, on the treatment of chronic phlebostatic ulcers. This study was conducted at the Department of Surgical Sciences of Rome's *Sapienza* University

The purpose of that study was to assess the efficacy of using dermic substitutes when treating patients suffering from chronic skin ailments due to and sustained by venous insufficiency.

This examination took parameters like the following into consideration: reduction of the size of the wound and improvement of quality of life (QoL) as expressed subjectively, on the basis

The study sample involved 13 patients suffering from chronic venous insufficiency (CVI) and postthrombotic syndrome, 5 of whom were also affected by pronounced varicosity, with incontinence of the saphenous-femoral junction and protracted reflux of the great saphenous vein (GSV). Ten of the patients, before proceeding to treatment with dermal substitutes, had been treated surgically for phlebopathy: six had undergone "stripping" of the GSV (of the "short" in two cases); two patients underwent crossectomy due to recurrence accompanied by inguinalcrural cavernoma; in five of the patients, the "feeder" veins were identified and linked, two of them as treatment in isolation (the other three in association with treatment for varices); and in the remaining three cases, the only management, besides local intervention, was elastic

Eight other patients, initially included in the study, were excluded later on because of the impossibility of proceeding with the therapy in the manner set down by the team's protocol.

• They had to present with chronic venous insufficiency, accompanied or not by varicosity

.

The patients were chosen according to the following criteria:

• Their ulcers had to have an area no greater than 20 cm2

their ongoing management.

of a number of elective factors.

**2.1. Materials and methodology**

• They had to be aged 50 or over.

of the great saphenous vein.

compression.

between October 2016 and March 2018.

**Figure 2.** Flow diagram showing the optimization and standardisation phases to isolate and expand in vitro ASCs derived from the mediastinal fat depots.

**Figure 3.** Optical image of ASCs at passage 3 cultured in PL and displaying the typically spindle-shaped morphology (A). Note that platelet lysate is able to preserve the mesodermal transdifferentiation of ASCs towards the adipogenic (B), osteogenic (C) and chondrogenic (D) lineages. Magnification 5×.

in vitro culturing of skin substitutes exists already, providing the treatment of a wide range of dermal disorders, such as burns, chronic ulcers, giant congenital melanocytic nevi and even the reconstruction of epithelial mucosa [53–58]. Specifically, the epithelial "organoid" developed by our research group is based on a combination of transplanted autologous cells seeded in biomimetic scaffolds. This methodology has been successfully established, is clinically available at several hospitals collaborating with *Sapienza* and has already been shown to significantly reduce hospitalisation time and costs [59].

Our group's experience of advanced dressings and dermal substitutes over the years during treatment of patients with acute and chronic ulcers of multifactorial origin (arteriopathic, phlebopathic, immunological and traumatic) produced a study based on chronic arteriopathic patients, the results of which were published in the article "Wounds Difficult to Heal: An Effective Treatment Strategy" [60]. There we highlighted the fact that recognition of the aetiology of a skin lesion and the correction of the pathophysiological conditions that determine and support it are the assumption and "step" fundamental to the success of local treatment. It also emerged that a "standard", univocal treatment applicable to all and every kind of wound does not exist. Appropriate local treatment involves a combination of multiple medications, products and devices demanding respect of their timing and guarantees regarding their ongoing management.

In a very recent study we focused, instead, on the treatment of chronic phlebostatic ulcers. This study was conducted at the Department of Surgical Sciences of Rome's *Sapienza* University between October 2016 and March 2018.

The purpose of that study was to assess the efficacy of using dermic substitutes when treating patients suffering from chronic skin ailments due to and sustained by venous insufficiency.

This examination took parameters like the following into consideration: reduction of the size of the wound and improvement of quality of life (QoL) as expressed subjectively, on the basis of a number of elective factors.

### **2.1. Materials and methodology**

The study sample involved 13 patients suffering from chronic venous insufficiency (CVI) and postthrombotic syndrome, 5 of whom were also affected by pronounced varicosity, with incontinence of the saphenous-femoral junction and protracted reflux of the great saphenous vein (GSV). Ten of the patients, before proceeding to treatment with dermal substitutes, had been treated surgically for phlebopathy: six had undergone "stripping" of the GSV (of the "short" in two cases); two patients underwent crossectomy due to recurrence accompanied by inguinalcrural cavernoma; in five of the patients, the "feeder" veins were identified and linked, two of them as treatment in isolation (the other three in association with treatment for varices); and in the remaining three cases, the only management, besides local intervention, was elastic compression.

Eight other patients, initially included in the study, were excluded later on because of the impossibility of proceeding with the therapy in the manner set down by the team's protocol.

The patients were chosen according to the following criteria:

• They had to be aged 50 or over.

in vitro culturing of skin substitutes exists already, providing the treatment of a wide range of dermal disorders, such as burns, chronic ulcers, giant congenital melanocytic nevi and even the reconstruction of epithelial mucosa [53–58]. Specifically, the epithelial "organoid" developed by our research group is based on a combination of transplanted autologous cells seeded in biomimetic scaffolds. This methodology has been successfully established, is clinically available at several hospitals collaborating with *Sapienza* and has already been shown

**Figure 3.** Optical image of ASCs at passage 3 cultured in PL and displaying the typically spindle-shaped morphology (A). Note that platelet lysate is able to preserve the mesodermal transdifferentiation of ASCs towards the adipogenic (B),

**Figure 2.** Flow diagram showing the optimization and standardisation phases to isolate and expand in vitro ASCs

to significantly reduce hospitalisation time and costs [59].

osteogenic (C) and chondrogenic (D) lineages. Magnification 5×.

derived from the mediastinal fat depots.

222 Wound Healing - Current Perspectives


The presence of undermined margins was an indication of treatment with infiltrations of platelet concentrate (PC) or mesenchymal ("regenerate") cells.

The platelet concentrate should be 1 × 10<sup>6</sup>

centration should always be 1 × 10<sup>6</sup>

described above.

foreseen.

microbiological test to verify their sterile state.

umbilical cord or fractional blood from an adult donor is used.

compatible with the blood group of the candidate for treatment are used.

the ulcer, making sure that it covers the entire area of the lesion.

of 3 weeks), Thiersch thin dermo-epidermal grafts were carried out.

/ml ± 20%. All the preparations thus obtained are

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Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

/ml ± 20%. The CP thus obtained is stored in the manner

checked for the biological validation required by law; a blood count is carried out as well as a

To keep the CP, sterile containers are used, each dose is rendered identifiable by its donation code; the type of the blood component and the expiry date of the product are also provided. All production and packaging procedures are carried out in aseptic conditions, in a sterilewelded closed circuit or under a laminar flow hood during the phases when it is necessary to open the circuit. The pouches containing the CP are deposited in protective cases with labels bearing the same data as those indicated on each dose and stored in a freezer at −80°C.

In cases where the patient's clinical and/or haematological conditions, the size of the ulcer and the number of medications to be carried out do not permit harvesting of autologous blood,

To produce CP from umbilical-cord blood, cord blood units deemed unsuitable for the haematopoietic stem-cell transplant bank are availed of. Cord units are considered suitable for the production of CP if they meet the foreseen regulatory requirements and are endorsed by specific informed consent as prescribed for their use. The production method used is the same as that described for CP obtained from whole autologous blood. For the treatment units

For the production of homologous fractional-blood CP, the blood component is prepared by collecting one or more units of single buffy-coat platelets and a bag of freshly frozen plasma, fractioned with blood donations free of transmissible viral diseases. The final platelet con-

On the day of the treatment, the platelet concentrate is defrosted and activated by adding 10% calcium gluconate (0.3 ml of Ca per ml of CP): in practice, 10 ml of the CP are placed on a sterile plate and mixed with 3 ml of calcium gluconate. The solution, stirred gently and left to rest for 10′–15′, forms the platelet gel to be placed on a sterile gauze and placed directly on

CP gel was used on seven patients, in compliance with our protocol of one application every 4 days for a maximum of 8 weeks (for a total of 14 dressings), in three cases homologous CP (with low Hb values found upon haemochromocytometric examination), and in four cases, autologous CPs (blood sampling of 410 cc) were considered sufficient for the applications

In six patients, the "regenerate" system was used, with one application every 4–5 days, for a maximum of four applications, over a total of about 20 days; in three of these cases, the treatment was used in association with and subsequent to CP, because it had not been possible to prolong the application of CP for the entire 8-week period (in the case of two patients) or because, at the end of the maximum number of 14 applications of the platelet concentrate, a completely unsatisfactory result was obtained (in the case of one patient); in the other three cases, after treatment with Rigenera (for a maximum of four applications over a total period

The presence of wound contamination was a criterion dictating temporary exclusion although patients were readmitted to the study once this condition was resolved.

All the patients provided informed consent, having evaluated compliance with the proviso requiring their participation for the entire duration of the treatment.

The criteria for exclusion were:


All the patients were assessed preliminarily by an ecocolor Doppler examination and other tests useful for inclusion/exclusion in the study. The ultrasound check sought confirmation of proximal saphenous-femoral valve incontinence and/or of saphenous perforators; in cases with referred stripping, it looked for the presence of accessory saphenoids or lapses of the cross (cavernoma) and, as far as deep circulation was concerned, the patency and absence of reflux with severe incontinence. These data, together with an objective examination, were the criteria adopted for preliminary surgical treatment and for the choice of local treatment recurring to one or more of the four dermal substitutes foreseen by the study, in some cases in sequence and/or in combination.

All the patients underwent local "wound bed preparation", requiring different lengths of time for different patients. WBP was followed by the applications of dermal substitutes. The dermal substances used were autologous or homologous platelet concentrates (PC); "micrografts" of autologous mesenchymal origin ("Rigenera Activa" System); "HyaloMatrix" PA Tissue Reconstruction Matrix (Fidia), on a hyaluronic acid base; and "Integra" Dermal Regeneration Template (LifeSciences Corp.), on a collagen base.

For the preparation and application of platelet concentrate, both from whole autologous and homologous blood, a specific protocol is followed: in the former case, the donor's personal data are recorded in the Blood Transfusion Service's management system (Emonet) which assigns an identification number to the donation; the blood collected is contained in a bag containing ACD (citrate glucose) anticoagulant, and the CP is produced by recurring to two centrifugation cycles: the first of these at a low rpm rate (210 g × 10 minutes) to obtain plateletrich plasma and eliminate the red blood cells and leucocytes and the second at a higher rpm rate (2000 g × 15 minutes) to concentrate the platelets by eliminating the depleted plasma. The platelet concentrate should be 1 × 10<sup>6</sup> /ml ± 20%. All the preparations thus obtained are checked for the biological validation required by law; a blood count is carried out as well as a microbiological test to verify their sterile state.

The presence of undermined margins was an indication of treatment with infiltrations of

The presence of wound contamination was a criterion dictating temporary exclusion although

All the patients provided informed consent, having evaluated compliance with the proviso

• The use of anti-inflammatory drugs, immunosuppressant and cytostatic drugs and oral anticoagulants for severe comorbidity (chronic renal failure requiring dialysis, congestive cardiomyopathies, liver failure) and concomitant arteriopathies (mixed genesis of the ulcer) • Poor/low self-sufficiency and/or lack of family or caregiving support, a factor mandatory

All the patients were assessed preliminarily by an ecocolor Doppler examination and other tests useful for inclusion/exclusion in the study. The ultrasound check sought confirmation of proximal saphenous-femoral valve incontinence and/or of saphenous perforators; in cases with referred stripping, it looked for the presence of accessory saphenoids or lapses of the cross (cavernoma) and, as far as deep circulation was concerned, the patency and absence of reflux with severe incontinence. These data, together with an objective examination, were the criteria adopted for preliminary surgical treatment and for the choice of local treatment recurring to one or more of the four dermal substitutes foreseen by the study, in some cases

All the patients underwent local "wound bed preparation", requiring different lengths of time for different patients. WBP was followed by the applications of dermal substitutes. The dermal substances used were autologous or homologous platelet concentrates (PC); "micrografts" of autologous mesenchymal origin ("Rigenera Activa" System); "HyaloMatrix" PA Tissue Reconstruction Matrix (Fidia), on a hyaluronic acid base; and "Integra" Dermal

For the preparation and application of platelet concentrate, both from whole autologous and homologous blood, a specific protocol is followed: in the former case, the donor's personal data are recorded in the Blood Transfusion Service's management system (Emonet) which assigns an identification number to the donation; the blood collected is contained in a bag containing ACD (citrate glucose) anticoagulant, and the CP is produced by recurring to two centrifugation cycles: the first of these at a low rpm rate (210 g × 10 minutes) to obtain plateletrich plasma and eliminate the red blood cells and leucocytes and the second at a higher rpm rate (2000 g × 15 minutes) to concentrate the platelets by eliminating the depleted plasma.

platelet concentrate (PC) or mesenchymal ("regenerate") cells.

patients were readmitted to the study once this condition was resolved.

requiring their participation for the entire duration of the treatment.

• Exposure of bone or nerve, ligament or aponeurotic tissue

Regeneration Template (LifeSciences Corp.), on a collagen base.

• The presence of immunohaematological disease • Neoplasms and chemoradiotherapy treatment

The criteria for exclusion were:

224 Wound Healing - Current Perspectives

for participation in the study

in sequence and/or in combination.

To keep the CP, sterile containers are used, each dose is rendered identifiable by its donation code; the type of the blood component and the expiry date of the product are also provided. All production and packaging procedures are carried out in aseptic conditions, in a sterilewelded closed circuit or under a laminar flow hood during the phases when it is necessary to open the circuit. The pouches containing the CP are deposited in protective cases with labels bearing the same data as those indicated on each dose and stored in a freezer at −80°C.

In cases where the patient's clinical and/or haematological conditions, the size of the ulcer and the number of medications to be carried out do not permit harvesting of autologous blood, umbilical cord or fractional blood from an adult donor is used.

To produce CP from umbilical-cord blood, cord blood units deemed unsuitable for the haematopoietic stem-cell transplant bank are availed of. Cord units are considered suitable for the production of CP if they meet the foreseen regulatory requirements and are endorsed by specific informed consent as prescribed for their use. The production method used is the same as that described for CP obtained from whole autologous blood. For the treatment units compatible with the blood group of the candidate for treatment are used.

For the production of homologous fractional-blood CP, the blood component is prepared by collecting one or more units of single buffy-coat platelets and a bag of freshly frozen plasma, fractioned with blood donations free of transmissible viral diseases. The final platelet concentration should always be 1 × 10<sup>6</sup> /ml ± 20%. The CP thus obtained is stored in the manner described above.

On the day of the treatment, the platelet concentrate is defrosted and activated by adding 10% calcium gluconate (0.3 ml of Ca per ml of CP): in practice, 10 ml of the CP are placed on a sterile plate and mixed with 3 ml of calcium gluconate. The solution, stirred gently and left to rest for 10′–15′, forms the platelet gel to be placed on a sterile gauze and placed directly on the ulcer, making sure that it covers the entire area of the lesion.

CP gel was used on seven patients, in compliance with our protocol of one application every 4 days for a maximum of 8 weeks (for a total of 14 dressings), in three cases homologous CP (with low Hb values found upon haemochromocytometric examination), and in four cases, autologous CPs (blood sampling of 410 cc) were considered sufficient for the applications foreseen.

In six patients, the "regenerate" system was used, with one application every 4–5 days, for a maximum of four applications, over a total of about 20 days; in three of these cases, the treatment was used in association with and subsequent to CP, because it had not been possible to prolong the application of CP for the entire 8-week period (in the case of two patients) or because, at the end of the maximum number of 14 applications of the platelet concentrate, a completely unsatisfactory result was obtained (in the case of one patient); in the other three cases, after treatment with Rigenera (for a maximum of four applications over a total period of 3 weeks), Thiersch thin dermo-epidermal grafts were carried out.

The "Hyalomatrix PA" dermal substitute was applied to one patient and the "Integra" dermal substitute to two patients. This choice was made when the patients presented a particularly "lively" tissue granulation phase and the size of the lesion was close to the 20-cm2 limit (the size of the product used being 5 × 5 cm). These patients were medicated every 3–4 days, according to the modalities set down in the technical data sheet, until the product was absorbed. On the basis of our protocol, all 13 patients were medicated every 3–7 days, monitored and observed for a total period of 8 weeks.

At the beginning of the study and at the end of the 8-week treatment period, in order to assess its efficacy, the following parameters were considered and used to define the results:


All these are elements that strongly impact upon life relationships and recovery of habitual daily activities, including work. A numerical value was attributed to each parameter, used together with all the others and employed to calculate overall average values.

Our study's 13-patient sample, as shown in **Table 1**, included 6 females and 7 males, whose ages ranged between 65 and 77 (for a mean age of 71); at the beginning of the treatment, the maximum average diameter of the wounds was 5 cm, a range of between 3 and 6.5 cm.

> a 60% reduction, a result just marginally better than the previous one; but one needs to keep in mind that these presented graver lesions are harder to manage than those of other patients. The three other cases, treated with Rigenera and a Thiersch graft, achieved a 55% reduction in the ulcer's greatest diameter. The two patients treated with the "Integra" dermic substitute obtained a 52.5% diminution of the wound's maximum diameter. Finally, the patient treated with the "Hyalomatrix" skin substitute achieved a 60% reduction. On the whole, by the end of

**Time 0 4 weeks 8 weeks P-value**

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

**Clearly ameliorated (D)**

227

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All the patients during the period of treatment reported a gradual reduction in pain, from an initial average of 7 on the NRS scale (range 9–5) to an average of 3 (range 6–2) at the end of the period (see graph 1). On the whole, the average reduction of perceived pain dropped by 4

At the end of the 8-week treatment period, an average of 8.25 out of 13 patients reported improvements in their quality of life, a spontaneous reduction in pain, a lessening of itchiness and secretion, lower incidence of bad odour with a recovery of functional capacity and almost complete resumption of habitual everyday activities. The results are summarised in **Tables 1**–**3**. It needs to be pointed out that during the treatment period no superinfections of the wounds requiring interruption of the therapy arose nor did other secondary complications associated

the observation period, the average reduction of the lesion for 13 patients was 57%.

Average value 6.9 4.3 3.2 0.001

Nocturnal rest 0 pts. 3 pts. 3 pts. 7 pts. Itch 0 pts. 3 pts. 2 pts. 8 pts. Pain 0 pts. 0 pts. 4 pts. 9 pts. Need of analgesics 0 pts. 0 pts. 3 pts. 10 pts. Mobilisation 0 pts. 3 pts. 3 pts. 7 pts. Foul odour 0 pts. 2 pts. 2 pts. 9 pts. Restart of daily activities 0 pts. 2 pts. 4 pts. 7 pts. Social and relational life 0 pts. 1 pts. 3 pts. 9 pts. Total (average values) 0 1.75 3 8.25

**Aggravated (A) Unmodified (B) Slightly ameliorated** 

**(C)**

Standard deviation 1.3 1.3 1.2

**Table 2.** Variations of the "pain" from the beginning to the end of the treatment.

points on the NRS numerical scale.

**Table 3.** Variations in QoL, stated by the patients.

**Pain (numerical rating scale)**

with the use of the products occur.

### **2.2. Results**

During the 8 weeks of treatment, some signs of improvement were observed in all patients: appearance in the wound bed of small islands of granulation tissue, superficialization of the bottom of the ulcer and growth of margin tissue. During the first 30 days, a reduction in over 25% of the area of the wound was observed; a reduction in more than 50% was observed in the 10 patients who underwent preliminary surgical treatment. In the remaining three cases, that is, the patients subjected to elastic-compressive bandaging only, there was an average reduction in 45% by the end of the 8 week.

The first four patients treated with CP, obtained on average a 57.5% reduction of the wound's maximum diameter. The three patients treated with a combination of CP and Rigenera achieved


**Table 1.** Variations of the diameters of the lesions from the beginning to the end of the treatment.


**Table 2.** Variations of the "pain" from the beginning to the end of the treatment.


**Table 3.** Variations in QoL, stated by the patients.

The "Hyalomatrix PA" dermal substitute was applied to one patient and the "Integra" dermal substitute to two patients. This choice was made when the patients presented a particularly

of the product used being 5 × 5 cm). These patients were medicated every 3–4 days, according to the modalities set down in the technical data sheet, until the product was absorbed. On the basis of our protocol, all 13 patients were medicated every 3–7 days, monitored and observed

At the beginning of the study and at the end of the 8-week treatment period, in order to assess

**b.** Patients' subjective perception of pain according to the NRS numerical scale, a scale of values from 0 to 10, where 0 corresponds to absence of pain and 10 to the maximum level

**c.** Quality of life, this too based on patients' subjective judgement, with reference to nighttime rest, itching, pain, need for painkillers, wet impregnation of the wound, bad smell

All these are elements that strongly impact upon life relationships and recovery of habitual daily activities, including work. A numerical value was attributed to each parameter, used

Our study's 13-patient sample, as shown in **Table 1**, included 6 females and 7 males, whose ages ranged between 65 and 77 (for a mean age of 71); at the beginning of the treatment, the maximum average diameter of the wounds was 5 cm, a range of between 3 and 6.5 cm.

During the 8 weeks of treatment, some signs of improvement were observed in all patients: appearance in the wound bed of small islands of granulation tissue, superficialization of the bottom of the ulcer and growth of margin tissue. During the first 30 days, a reduction in over 25% of the area of the wound was observed; a reduction in more than 50% was observed in the 10 patients who underwent preliminary surgical treatment. In the remaining three cases, that is, the patients subjected to elastic-compressive bandaging only, there was an average

The first four patients treated with CP, obtained on average a 57.5% reduction of the wound's maximum diameter. The three patients treated with a combination of CP and Rigenera achieved

Average value 5.0 2.1 56.9 0.001

Standard deviation 1.1 0.6 7.2

**Table 1.** Variations of the diameters of the lesions from the beginning to the end of the treatment.

**Beginning (cm) End (cm) Reduction (%) P-value**

together with all the others and employed to calculate overall average values.

its efficacy, the following parameters were considered and used to define the results:

limit (the size

"lively" tissue granulation phase and the size of the lesion was close to the 20-cm2

for a total period of 8 weeks.

226 Wound Healing - Current Perspectives

of pain perceived

**2.2. Results**

and hygiene of the wound.

reduction in 45% by the end of the 8 week.

**Diameters of the ulcers during the treatment**

**a.** The extent of the reduction of the size of the ulcer

a 60% reduction, a result just marginally better than the previous one; but one needs to keep in mind that these presented graver lesions are harder to manage than those of other patients. The three other cases, treated with Rigenera and a Thiersch graft, achieved a 55% reduction in the ulcer's greatest diameter. The two patients treated with the "Integra" dermic substitute obtained a 52.5% diminution of the wound's maximum diameter. Finally, the patient treated with the "Hyalomatrix" skin substitute achieved a 60% reduction. On the whole, by the end of the observation period, the average reduction of the lesion for 13 patients was 57%.

All the patients during the period of treatment reported a gradual reduction in pain, from an initial average of 7 on the NRS scale (range 9–5) to an average of 3 (range 6–2) at the end of the period (see graph 1). On the whole, the average reduction of perceived pain dropped by 4 points on the NRS numerical scale.

At the end of the 8-week treatment period, an average of 8.25 out of 13 patients reported improvements in their quality of life, a spontaneous reduction in pain, a lessening of itchiness and secretion, lower incidence of bad odour with a recovery of functional capacity and almost complete resumption of habitual everyday activities. The results are summarised in **Tables 1**–**3**.

It needs to be pointed out that during the treatment period no superinfections of the wounds requiring interruption of the therapy arose nor did other secondary complications associated with the use of the products occur.

### **3. Perspectives and conclusions**

Despite advances in wound-healing treatment, dermal tissue still remains a difficult organ to regenerate. Our work in the future will probably consist in multistep approaches rather than in single repair strategies, which have proven to be only partially efficacious. Future strategies will, most likely, combine stem-cell properties, next-generation scaffolds or vehicles (i.e. nanoparticles) and growth factors or supplements, like PL. Improvements in our understanding of skin biology and the physiological processes of wound repair should permit us to interpret healing microenvironments better. To achieve our final goal, we will be required to design more personalised therapies, taking into account genetic variability, wound types as well as patients' clinical and metabolic features.

• The use of these substitutes *does* cause a reduction in the size of ulcers, improving, above all, the quality of life of patients. One notices, in particular, a reduction in levels of pain and

Multidisciplinary Approaches to the Stimulation of Wound Healing and Use of Dermal…

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229

• The choice of the best therapy, from among the different options available, also depends on the physician's ability to perceive the one most suitable for the type and characteristics

In conclusion, the numerous technological opportunities made available by regenerative medicine, including advanced dressings and dermal substitutes, if used correctly and following a multidisciplinary approach if necessary, seem to offer advantages in terms of clinical efficacy, patients' quality of life and, last but not least, also in terms of healthcare costs.

resumption of habitual everyday activities.

**Acknowledgements**

**Conflicts of interest**

**Author details**

Raffaele Capoano1

Lidia De Felice3

Rome, Italy

**References**

Therapy. 2015;**6**:241

The authors declare no conflicts of interest.

\*, Rita Businaro<sup>2</sup>

\*Address all correspondence to: raffaele.capoano@uniroma1.it

1 Department of Surgical Sciences, "Sapienza" University of Rome, Italy

3 Department of Molecular Medicine, "Sapienza" University of Rome, Italy

and Bruno Salvati<sup>1</sup>

of the patient and the availability of his/her caregivers.

This work was supported by Ateneo ("Sapienza" University of Rome).

, Besar Kolce<sup>1</sup>

2 Department of Medico-Surgical Sciences and Biotechnologies, "Sapienza" University of

[1] Rodriguez J, Boucher F, Lequeux C, et al. Intradermal injection of human adiposederived stem cells accelerates skin wound healing in nude mice. Stem Cell Research &

, Andrea Biancucci<sup>1</sup>

, Silvia Izzo<sup>1</sup>

,

It is not possible, in actual fact, to reach definitive conclusions given the variability of the factors capable of influencing the outcome of therapy and the difficulty of rendering populations of patients treated totally homogeneous. Furthermore, this aspect also emerges from the field's latest literature [60–62], being the only source available at present (since there are no definitive guidelines available). In addition, this kind of patient is not always willing to follow the lengthy periods of treatment often deemed necessary or comply with the temporal parameters the cure requires. Due to certain events like intercurrent pathologies, hospitalisation or logistic problems, exclusion from a study sample may become inevitable. For these reasons we consider the results obtained here as preliminary.


In conclusion, the numerous technological opportunities made available by regenerative medicine, including advanced dressings and dermal substitutes, if used correctly and following a multidisciplinary approach if necessary, seem to offer advantages in terms of clinical efficacy, patients' quality of life and, last but not least, also in terms of healthcare costs.

### **Acknowledgements**

**3. Perspectives and conclusions**

228 Wound Healing - Current Perspectives

well as patients' clinical and metabolic features.

treatment, jeopardising the outcome.

to all the developmental stages of an ulcer.

reasons we consider the results obtained here as preliminary.

Despite advances in wound-healing treatment, dermal tissue still remains a difficult organ to regenerate. Our work in the future will probably consist in multistep approaches rather than in single repair strategies, which have proven to be only partially efficacious. Future strategies will, most likely, combine stem-cell properties, next-generation scaffolds or vehicles (i.e. nanoparticles) and growth factors or supplements, like PL. Improvements in our understanding of skin biology and the physiological processes of wound repair should permit us to interpret healing microenvironments better. To achieve our final goal, we will be required to design more personalised therapies, taking into account genetic variability, wound types as

It is not possible, in actual fact, to reach definitive conclusions given the variability of the factors capable of influencing the outcome of therapy and the difficulty of rendering populations of patients treated totally homogeneous. Furthermore, this aspect also emerges from the field's latest literature [60–62], being the only source available at present (since there are no definitive guidelines available). In addition, this kind of patient is not always willing to follow the lengthy periods of treatment often deemed necessary or comply with the temporal parameters the cure requires. Due to certain events like intercurrent pathologies, hospitalisation or logistic problems, exclusion from a study sample may become inevitable. For these

• Having said this, we are in a position to assert that skin substitutes are capable of determining a clinical improvement of chronic ulcers caused by pathologies of the vein when, after use of traditional medication, or accurate advanced debridement, the condition of the ulcer is such that regeneration of tissue is feasible. Indeed, timing seems essential for prescriptions as well: both precocious and tardy dressing may nullify the effectiveness of a

• The integrated use of different compounds can prove useful, especially in relation to the long periods often needed to obtain complete healing. Every single treatment may determine partial, even substantial, improvement but may fail to cure the lesion completely: there is no "ideal" medication for all ulcers, just as there is no "ideal" medication applicable

• The specific treatment of comorbidities and restoration of a satisfactory level of nutrition are of great importance when pursuing positive outcomes of any local therapy, though chances of complete success are related mainly to a correct diagnosis regarding the origin of the ulcer and, above all, the removal, surgical if necessary, of factors impeding recovery: "The beginning of healing lies in knowledge of the ailment" (*Epicurus, 341–270 BCE*). • Dermal substitutes have become, therefore, part of the modern concept of the multidisciplinary approach to the treatment of chronic skin lesions, in particular, the management of wounds that are less likely to heal availing of standard therapy. They represent a valid therapeutic "step", whether used alone or in combination, also considering the potential clinical benefits demonstrated and the low percentage of complications related to their use.

This work was supported by Ateneo ("Sapienza" University of Rome).

### **Conflicts of interest**

The authors declare no conflicts of interest.

### **Author details**

Raffaele Capoano1 \*, Rita Businaro<sup>2</sup> , Besar Kolce<sup>1</sup> , Andrea Biancucci<sup>1</sup> , Silvia Izzo<sup>1</sup> , Lidia De Felice3 and Bruno Salvati<sup>1</sup>

\*Address all correspondence to: raffaele.capoano@uniroma1.it

1 Department of Surgical Sciences, "Sapienza" University of Rome, Italy

2 Department of Medico-Surgical Sciences and Biotechnologies, "Sapienza" University of Rome, Italy

3 Department of Molecular Medicine, "Sapienza" University of Rome, Italy

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**Chapter 14**

**Provisional chapter**

**The Impact of Biofilm Formation on Wound Healing**

Chronic wounds represent an important challenge for wound care and are universally colonized by bacteria. These bacteria can form biofilm as a survival mechanism that confers the ability to resist environmental stressors and antimicrobials due to a variety of reasons, including low metabolic activity. Additionally, the exopolymeric substance (EPS) contained in biofilm acts as a mechanical barrier to immune system cells, leading to collateral damage in the surrounding tissue as well as chronic inflammation, which eventually will delay healing of the wound. This chapter will discuss current knowledge on biofilm formation, its presence in acute and chronic wounds, how biofilm affects antibiotic resistance and tolerance, as well as the wound healing process. We will also discuss proposed methods to eliminate biofilm and improve wound healing despite its presence,

**Keywords:** biofilm, chronic wounds, delayed healing, exopolymeric substance, slime,

Intact skin provides a protective barrier to bacterial invasion. Any wound comprises a break

Along with hypoxia/poor perfusion, ischemia-reperfusion injury, and inadequate offloading or compression therapy, microbial infection is one of the most significant causes of delay in

Over the last few years, bacterial biofilms in general and their role in chronic wounds have been the subject of intense research. Biofilms have been reported to be present in 60% [4] to

including basic science and clinical studies regarding these matters.

in this epidermal barrier, allowing microbial invasion into deeper layers.

**The Impact of Biofilm Formation on Wound Healing**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.85020

Rafael A. Mendoza, Ji-Cheng Hsieh and

Rafael A. Mendoza, Ji-Cheng Hsieh

http://dx.doi.org/10.5772/intechopen.85020

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Robert D. Galiano

**Abstract**

extracellular matrix

**1. Introduction**

healing [1–3].

and Robert D. Galiano


#### **The Impact of Biofilm Formation on Wound Healing The Impact of Biofilm Formation on Wound Healing**

DOI: 10.5772/intechopen.85020

Rafael A. Mendoza, Ji-Cheng Hsieh and Robert D. Galiano Rafael A. Mendoza, Ji-Cheng Hsieh and Robert D. Galiano

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.85020

#### **Abstract**

[59] Businaro R, Corsi M, Di Raimo T, Marasco S, Laskin DL, Salvati B, et al. Multidisciplinary approaches to stimulate wound healing. Annals of the New York Academy of Sciences.

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[62] Capoano R, Businaro R, Tesori MC, Donello C, Lombardo F, Lo Vasco VR, et al. Wounds difficult to heal: An effective treatment strategy. Current Vascular Pharmacology. 2017;

gel: A prospective case series. Ostomy/Wound Management. 2010;**56**:36-44

2016;**1378**(1):137-142

234 Wound Healing - Current Perspectives

**15**(6):582-588

Chronic wounds represent an important challenge for wound care and are universally colonized by bacteria. These bacteria can form biofilm as a survival mechanism that confers the ability to resist environmental stressors and antimicrobials due to a variety of reasons, including low metabolic activity. Additionally, the exopolymeric substance (EPS) contained in biofilm acts as a mechanical barrier to immune system cells, leading to collateral damage in the surrounding tissue as well as chronic inflammation, which eventually will delay healing of the wound. This chapter will discuss current knowledge on biofilm formation, its presence in acute and chronic wounds, how biofilm affects antibiotic resistance and tolerance, as well as the wound healing process. We will also discuss proposed methods to eliminate biofilm and improve wound healing despite its presence, including basic science and clinical studies regarding these matters.

**Keywords:** biofilm, chronic wounds, delayed healing, exopolymeric substance, slime, extracellular matrix

**1. Introduction**

Intact skin provides a protective barrier to bacterial invasion. Any wound comprises a break in this epidermal barrier, allowing microbial invasion into deeper layers.

Along with hypoxia/poor perfusion, ischemia-reperfusion injury, and inadequate offloading or compression therapy, microbial infection is one of the most significant causes of delay in healing [1–3].

Over the last few years, bacterial biofilms in general and their role in chronic wounds have been the subject of intense research. Biofilms have been reported to be present in 60% [4] to

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Representative scanning electron microscopy of wounds on mice dorsal skin. (A) Inoculated wound with *S. aureus* showed aggregates encased in EPS matrix. (B) Non-inoculated wound without EPS (image from Nguyen et al. [10], with permission).

associated with both acute and chronic wounds *via* different rates and mechanisms. An infection with a more predominantly planktonic phenotype is more aggressive, with rapidly dividing cells invading host tissues and stimulating a strong inflammatory response typical of an acute infection. Several microorganisms can adopt a different, sessile phenotype, called a biofilm, that allows them to attach to biotic or abiotic surfaces, form aggregates, and regulate the production of an extracellular polymeric substance (EPS), contributing to their ability to

**Table 1.** Planktonic and biofilm phenotypes comparison in regards to various bacterial traits and behaviors.

**Trait Planktonic Biofilm**

Risk for antibiotic resistance Moderate High Spread Disseminating Sessile Extracellular polymetric substance (EPS) No Yes Metabolic activity High Low Species count Monospecies, polyspecies Polyspecies

Virulence Acute, aggressive course Chronic disease Host inflammatory response High, sudden Mild, persistent

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 237

This aggregate or cluster, once called "*slime*," constitutes the biofilm, a complex tertiary structure of sessile communities of one or more species of bacteria embedded within a matrix of EPS. The EPS is composed mainly by water, polysaccharides, DNA and other substances secreted by the embedded bacteria, but also by substances scavenged from the host. It is important to appreciate that all the building blocks of a wound biofilm are ultimately derived from the wound bed and skin. Cell lysis and subsequent local decomposition of the EPS matrix is advantageous for the biofilm population, creating new pores and channels that improve nutrient access, and the intracellular level of the second messenger cyclic di-GMP are involved in regulating biofilm formation and the production of matrix

For many years, biofilm has been known to exist on dental plaque and industrial water processing and even considered the predominant state of bacteria within the human body [16]. Later on, its presence was reported on endocardium, urinary tract mucosa, nasal and sinus epithelium and pulmonary tissue, and more recently biofilms have been found in healed surgical wounds, sutures, implants and IV catheters which can be contaminated at time of insertion or as a result of hematologic seeding from a colonized tissue. The relationship between biofilm and host will depend on the location and the bacterial composition of the biofilm; for instance, in the gastrointestinal mucosa, biofilm has a commensal behavior, while in wounds or respiratory tract mucosa, a pathogenic behavior. This difference is thought to be due to the

host's capacity to coexist or eradicate biofilm [6, 12, 17, 18].

survive [13, 14] (**Table 1**).

**Planktonic vs. biofilm phenotype**

components [15].

80% [5] of chronic wounds, and a recent meta-analysis confirms their presence in 78.2% of chronic wounds [2]. Therefore, biofilms have been categorized as an important factor in most chronic non-healing skin wounds [6].

Non-healing or poorly healing wounds affect close to 25 million people in the US [7], more than 7% of its population, while reports from the UK [8] predict that 1–2% of the population in developed countries will experience a chronic wound in their lifetime. Posnett et al. [9] reports the financial burden to the healthcare system of caring for chronic wounds in the UK, totaling US\$ 3.4–4.6 billion a year, close to 3% of the healthcare budget. The US, a larger and more complex system, observed \$35.3 billion in spending of Medicare funds on wound care alone in 2014, of which 16.7 billion was spent on infections and 9.4 billion on chronic ulcers [9] (**Figure 1**).

The implications of a biofilm-covered wound are not limited to delayed healing and financial burden. Biofilms pose a risk for persistent wound infections, especially when medical hardware is inserted into the body [11]. Biofilms can also develop into an overt infection, contribute to antimicrobial resistance, and increase the risk for adverse or tissue toxic effects caused by topical agents [12].

### **2. Background**

Wound healing can be deranged by multiple causes, including local hypoxia or poor perfusion, repetitive ischemia-reperfusion injury, inadequate offloading or compressive therapy, and bacterial infection. Bacterial infection, playing a great role among these causes, has been


**Planktonic vs. biofilm phenotype**

80% [5] of chronic wounds, and a recent meta-analysis confirms their presence in 78.2% of chronic wounds [2]. Therefore, biofilms have been categorized as an important factor in most

**Figure 1.** Representative scanning electron microscopy of wounds on mice dorsal skin. (A) Inoculated wound with *S. aureus* showed aggregates encased in EPS matrix. (B) Non-inoculated wound without EPS (image from Nguyen et al.

Non-healing or poorly healing wounds affect close to 25 million people in the US [7], more than 7% of its population, while reports from the UK [8] predict that 1–2% of the population in developed countries will experience a chronic wound in their lifetime. Posnett et al. [9] reports the financial burden to the healthcare system of caring for chronic wounds in the UK, totaling US\$ 3.4–4.6 billion a year, close to 3% of the healthcare budget. The US, a larger and more complex system, observed \$35.3 billion in spending of Medicare funds on wound care alone in 2014, of which 16.7 billion was spent on infections and 9.4 billion on chronic ulcers [9] (**Figure 1**).

The implications of a biofilm-covered wound are not limited to delayed healing and financial burden. Biofilms pose a risk for persistent wound infections, especially when medical hardware is inserted into the body [11]. Biofilms can also develop into an overt infection, contribute to antimicrobial resistance, and increase the risk for adverse or tissue toxic effects

Wound healing can be deranged by multiple causes, including local hypoxia or poor perfusion, repetitive ischemia-reperfusion injury, inadequate offloading or compressive therapy, and bacterial infection. Bacterial infection, playing a great role among these causes, has been

chronic non-healing skin wounds [6].

[10], with permission).

236 Wound Healing - Current Perspectives

caused by topical agents [12].

**2. Background**

**Table 1.** Planktonic and biofilm phenotypes comparison in regards to various bacterial traits and behaviors.

associated with both acute and chronic wounds *via* different rates and mechanisms. An infection with a more predominantly planktonic phenotype is more aggressive, with rapidly dividing cells invading host tissues and stimulating a strong inflammatory response typical of an acute infection. Several microorganisms can adopt a different, sessile phenotype, called a biofilm, that allows them to attach to biotic or abiotic surfaces, form aggregates, and regulate the production of an extracellular polymeric substance (EPS), contributing to their ability to survive [13, 14] (**Table 1**).

This aggregate or cluster, once called "*slime*," constitutes the biofilm, a complex tertiary structure of sessile communities of one or more species of bacteria embedded within a matrix of EPS. The EPS is composed mainly by water, polysaccharides, DNA and other substances secreted by the embedded bacteria, but also by substances scavenged from the host. It is important to appreciate that all the building blocks of a wound biofilm are ultimately derived from the wound bed and skin. Cell lysis and subsequent local decomposition of the EPS matrix is advantageous for the biofilm population, creating new pores and channels that improve nutrient access, and the intracellular level of the second messenger cyclic di-GMP are involved in regulating biofilm formation and the production of matrix components [15].

For many years, biofilm has been known to exist on dental plaque and industrial water processing and even considered the predominant state of bacteria within the human body [16]. Later on, its presence was reported on endocardium, urinary tract mucosa, nasal and sinus epithelium and pulmonary tissue, and more recently biofilms have been found in healed surgical wounds, sutures, implants and IV catheters which can be contaminated at time of insertion or as a result of hematologic seeding from a colonized tissue. The relationship between biofilm and host will depend on the location and the bacterial composition of the biofilm; for instance, in the gastrointestinal mucosa, biofilm has a commensal behavior, while in wounds or respiratory tract mucosa, a pathogenic behavior. This difference is thought to be due to the host's capacity to coexist or eradicate biofilm [6, 12, 17, 18].

### **3. Composition of biofilm**

Several functions have been attributed to biofilm: genetic material reservoir, nutrient source, matrix stabilization, adhesion, and bacterial communication. Most of these functions will depend on the particular substances present in the biofilm, which depend exclusively on the species and even the strain of the bacteria. For instance, *P. aeruginosa* produces a biofilm with a higher density EPS, with a well-defined matrix interspersed within clusters of bacterial cells and has the particularity to be predominant over other species in a polybacterial microbiome. In general, the interaction of these substances and how the bacteria inside the biofilm manage to utilize these substances will affect the morphology of the biofilm with common effects: immobilizing biofilm cells and allowing the existence of a very diverse habitat favoring biodiversity, where every member can contribute with their own EPS [15] (**Figure 2**).

biofilms. For instance, presence of Ca2+ in biofilm formed by mucoid strains of *P. aeruginosa* experienced an enhancement in their mechanical stability. In *S. epidermidis*, poly-*N*-acetylglu-

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 239

As seen in *P. aeruginosa* and *S. epidermidis*, polysaccharide compositions are very diverse, even between strains of a single species. *P. aeruginosa*, for instance, produces at least three distinct exopolysaccharides that have a direct effect on its biofilm architecture: alginate, *Pel* and *Psl*. Mucoid strains of *P. aeruginosa* contain alginate, an exopolysaccharide for biofilm formation that, although non-essential, has a notable effect on biofilm architecture. Alginate takes part at the beginning of biofilm formation and is responsible for the mechanical stability of mature biofilms. Alginate from this strain has a particular clinical relevance, being comprised of uronic acids, in that it can be used as an EPS marker, since this type of acid is not found inside the bacterial cells. In non-mucoid strains, *Pel* and *Psl* participate in the first stages of biofilm

Biofilms also contain a diversity of enzymes, lending a complex organization and capability of adaptation. Enzymes will break down biopolymers into low molecular mass products, degrade the structural EPS to promote detachment, act as virulence factors, and even degrade EPS components during starvation. Cell surface-associated proteins and extracellular carbohydrate-binding proteins (*lectins*) are also a key component in the biofilm, involved in the

Among these proteins we can find the glucan-binding proteins present in dental plaque caused by *S. mutans*, the galactose-specific lectin *lecA* and fucose-specific lectin *lecb* of *P. aeruginosa*, which have been associated with biofilm formation. Biofilm associated surface protein (*bap*) from *S. aureus* and the bap-like proteins, which promote biofilm formation in several species while also playing a role in bacterial infectious processes. Biofilms also contain amyloids, involved in adhesion to inanimate surfaces and host cells and invasion of host cells;

eDNA is an integral part of the matrix and biofilm mode of life. *B. cereus* uses DNA as an adhesion molecule, and in *P. aeruginosa*, eDNA serves as an intercellular connector, with DNase inhibiting biofilm formation specifically in *P. aeruginosa*. In *S. aureus*, eDNA serves the same structural role of PNAG in *S. epidermidis* eDNA, although seen initially as residual material from lysed cells, is also actively excreted. Although primarily occurring in waste-water biofilms, biofilms from various origins have been found to contain eDNA of varying levels and importance, even between closely related species. For example, eDNA plays a critical structural role in the biofilm matrix of *S. aureus* but only serves as a minor component of *S. epidermidis* biofilms. eDNA is localized differently between biofilms; in *P. aeruginosa*, for example, forms a grid-like structure. Additionally, eDNA has antimicrobial activity, having the ability to chelate cations that stabilize lipopolysaccharide and the bacterial outer mem-

additionally, they can function as cytotoxins for bacterial and plant cells [15].

cosamine (PNAG) makes a considerable contribution to biofilm integrity [15].

formation, while *Psl* alone is involved in adherence to surfaces [15].

formation and stabilization of the matrix network [15].

**3.2. Proteins**

**3.3. Extracellular DNA**

brane, provoking cell lysis [15].

#### **3.1. Polysaccharides**

Polysaccharides comprise a major fraction of the EPS matrix and are responsible for the biofilm's mechanical properties. Interestingly, it seems to be mainly the exopolysaccharides in multivalent inorganic ions with EPS can greatly influence the mechanical properties of

**Figure 2.** The extracellular polymeric substances matrix at different dimensions. (a) A model of a bacterial biofilm attached to a solid surface. (b) The major matrix components—polysaccharides, proteins and DNA—in a non-homogeneous pattern. (c) Physicochemical interactions and the entanglement of biopolymers that give stability to the EPS matrix. (d) A molecular modelling simulation of the interaction between alginate (right) and lipase (left) of *P. aeruginosa* (image from Flemming and Wingender [15], with permission).

biofilms. For instance, presence of Ca2+ in biofilm formed by mucoid strains of *P. aeruginosa* experienced an enhancement in their mechanical stability. In *S. epidermidis*, poly-*N*-acetylglucosamine (PNAG) makes a considerable contribution to biofilm integrity [15].

As seen in *P. aeruginosa* and *S. epidermidis*, polysaccharide compositions are very diverse, even between strains of a single species. *P. aeruginosa*, for instance, produces at least three distinct exopolysaccharides that have a direct effect on its biofilm architecture: alginate, *Pel* and *Psl*. Mucoid strains of *P. aeruginosa* contain alginate, an exopolysaccharide for biofilm formation that, although non-essential, has a notable effect on biofilm architecture. Alginate takes part at the beginning of biofilm formation and is responsible for the mechanical stability of mature biofilms. Alginate from this strain has a particular clinical relevance, being comprised of uronic acids, in that it can be used as an EPS marker, since this type of acid is not found inside the bacterial cells. In non-mucoid strains, *Pel* and *Psl* participate in the first stages of biofilm formation, while *Psl* alone is involved in adherence to surfaces [15].

### **3.2. Proteins**

Biofilms also contain a diversity of enzymes, lending a complex organization and capability of adaptation. Enzymes will break down biopolymers into low molecular mass products, degrade the structural EPS to promote detachment, act as virulence factors, and even degrade EPS components during starvation. Cell surface-associated proteins and extracellular carbohydrate-binding proteins (*lectins*) are also a key component in the biofilm, involved in the formation and stabilization of the matrix network [15].

Among these proteins we can find the glucan-binding proteins present in dental plaque caused by *S. mutans*, the galactose-specific lectin *lecA* and fucose-specific lectin *lecb* of *P. aeruginosa*, which have been associated with biofilm formation. Biofilm associated surface protein (*bap*) from *S. aureus* and the bap-like proteins, which promote biofilm formation in several species while also playing a role in bacterial infectious processes. Biofilms also contain amyloids, involved in adhesion to inanimate surfaces and host cells and invasion of host cells; additionally, they can function as cytotoxins for bacterial and plant cells [15].

#### **3.3. Extracellular DNA**

**Figure 2.** The extracellular polymeric substances matrix at different dimensions. (a) A model of a bacterial biofilm attached to a solid surface. (b) The major matrix components—polysaccharides, proteins and DNA—in a non-homogeneous pattern. (c) Physicochemical interactions and the entanglement of biopolymers that give stability to the EPS matrix. (d) A molecular modelling simulation of the interaction between alginate (right) and lipase (left) of *P. aeruginosa* (image from

Several functions have been attributed to biofilm: genetic material reservoir, nutrient source, matrix stabilization, adhesion, and bacterial communication. Most of these functions will depend on the particular substances present in the biofilm, which depend exclusively on the species and even the strain of the bacteria. For instance, *P. aeruginosa* produces a biofilm with a higher density EPS, with a well-defined matrix interspersed within clusters of bacterial cells and has the particularity to be predominant over other species in a polybacterial microbiome. In general, the interaction of these substances and how the bacteria inside the biofilm manage to utilize these substances will affect the morphology of the biofilm with common effects: immobilizing biofilm cells and allowing the existence of a very diverse habitat favoring biodi-

Polysaccharides comprise a major fraction of the EPS matrix and are responsible for the biofilm's mechanical properties. Interestingly, it seems to be mainly the exopolysaccharides in multivalent inorganic ions with EPS can greatly influence the mechanical properties of

versity, where every member can contribute with their own EPS [15] (**Figure 2**).

Flemming and Wingender [15], with permission).

**3. Composition of biofilm**

238 Wound Healing - Current Perspectives

**3.1. Polysaccharides**

eDNA is an integral part of the matrix and biofilm mode of life. *B. cereus* uses DNA as an adhesion molecule, and in *P. aeruginosa*, eDNA serves as an intercellular connector, with DNase inhibiting biofilm formation specifically in *P. aeruginosa*. In *S. aureus*, eDNA serves the same structural role of PNAG in *S. epidermidis* eDNA, although seen initially as residual material from lysed cells, is also actively excreted. Although primarily occurring in waste-water biofilms, biofilms from various origins have been found to contain eDNA of varying levels and importance, even between closely related species. For example, eDNA plays a critical structural role in the biofilm matrix of *S. aureus* but only serves as a minor component of *S. epidermidis* biofilms. eDNA is localized differently between biofilms; in *P. aeruginosa*, for example, forms a grid-like structure. Additionally, eDNA has antimicrobial activity, having the ability to chelate cations that stabilize lipopolysaccharide and the bacterial outer membrane, provoking cell lysis [15].


Biofilms play a significant role in the development of chronic cutaneous wounds, with up to 80% of chronic wounds having been found to contain a biofilm compared to 6% of acute

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 241

Biofilms cause chronic infections through mechanisms that are either innate or interact closely with the host immune system: genetic changes, surface and excreted molecular messengers, physical barriers, and escape behaviors. Although the bacteria may not disseminate throughout the body, pathogenicity is retained and arguably increased, as bacterial concentration within the biofilm increases and individuals tend to leave the biofilm, either through purposeful dissolution of EPS or through stresses on the biofilm itself by the fluid encasing the biofilm [19].

When bacteria cluster in a biofilm, movement of advantageous genetic traits, such as antibiotic resistance, throughout the constituents of the biofilm is expedited through transformation, horizontal gene transfer, or phage infection, making each individual bacterium even more virulent when it leaves the biofilm. *P. aeruginosa* biofilms, for example, exhibit a high concentration of DNA within their EPS. This also promotes significant genetic variability within a biofilm, increasing the chances that one of the many individuals will survive an environmental insult [19]. This includes antibiotics, leading to concern that excessive and inappropriate antibiotic use against biofilms expedites the development of antibiotic resistant strains [12].

Biofilms involve a complex relationship between bacteria virulence factors, survival mechanisms, and the host immune response [22]. Different species all exhibit particular biofilm characteristics that inhibit wound healing. EPS by itself represents a physical barrier against inflammatory cell phagocytosis, and has the potential to inhibit the complement cascade and antibiotic penetration into the wound [23]. Acellular extract from *S. aureus* biofilms inhibits the movement of keratinocytes and promotes apoptosis, leading to impaired cutaneous wound healing. This extract did not differ in pH or calcium levels; its effect on keratinocytes was due to direct cytotoxic substances secreted from or present on *S. aureus* bacterium: alpha-

*P. aeruginosa* biofilms similarly inhibit neutrophil movement but may spare their capacity for oxidative burst, and exhibit a capacity for ejecting individual bacterium from the biofilm [24]. Another potential mechanism for *P. aeruginosa* biofilm resistance to neutrophils is the rapid necrosis induced by the production of *rhamnolipids* [23]. Additionally, significant delay in wound healing, re-epithelization and collagen deposition have been reported without significant difference in PMN infiltration or granulation tissue [6]. The ultimate result is neutrophil aggregation near the biofilm, with oxidative burst products accumulating and causing neutrophil death, while individuals within the biofilm leave to create new colonies away from the initial site [24, 25].

Biofilms in general promote a host inflammatory response that poorly penetrates the biofilm itself, causing surrounding cell damage instead [18]. Host inflammatory signal expression also characterizes the biofilm infection; in general, those with impaired host immune responses, such as those with diabetes or arterial insufficiency, tend to have more significant

toxin and cell surface-expressed fibronectin-binding proteins [21].

wounds [2, 4, 5, 21].

**4.3. Wound healing inhibition**

wounds [22].

**Table 2.** Composition of biofilm exopolymeric substance (EPS) and associated functions.

#### **3.4 Water and biosurfactants**

Water is by far the largest component of the matrix, and water management is so critical that bacteria actively respond to desiccation by producing EPS. Molecular composition of the water component is critical as well, and the EPS matrix acts as a molecular sieve, sequestering cations, anions, nonpolar compounds and particles from the water phase. By comparison, biosurfactants have antibacterial and antifungal properties and are important for bacterial attachment and detachment from oil droplets. *Rhamnolipids*, which can act as surfactants, have been found in the EPS matrix of *P. aeruginosa* [15] (**Table 2**).

### **4. Pathophysiology**

#### **4.1. Biofilm development**

Biofilms utilize a variety of mechanisms in order to establish themselves. When exposed to adverse conditions, planktonic bacteria facilitate survival by forming biofilms. This occurs through "*phase variation*" and "*adaptive mutation*," genetic alterations that include point mutations, recombination, and transpositions, with the goal of producing individuals more capable of producing biofilms. *V. cholera*, *S. typhi*, and *E. coli* all exhibit stress-induced genetic alteration by adaptive mechanisms that produce a biofilm-capable phenotype, producing distinct, wrinkled individuals. *V. cholera* produces a more chlorine-resistant subtype called rugose, while *S. typhi* and *E. coli* change to an "*rdar*" phenotype, or red, dry, and rough [19].

#### **4.2. Biofilms and chronic disease**

By establishing biofilms, bacterial species not only increase their antibiotic resistance 1000-fold, they produce optimal conditions for chronic infections. By sacrificing aggressive movement throughout the body for confinement within a protective extracellular matrix, bacterial species effectively hide antigens, reduce the effectiveness of antibiotics, and blunt the immune response, promoting chronic disease: endocarditis, chronic kidney stones, and CF infections [19, 20]. Biofilms play a significant role in the development of chronic cutaneous wounds, with up to 80% of chronic wounds having been found to contain a biofilm compared to 6% of acute wounds [2, 4, 5, 21].

Biofilms cause chronic infections through mechanisms that are either innate or interact closely with the host immune system: genetic changes, surface and excreted molecular messengers, physical barriers, and escape behaviors. Although the bacteria may not disseminate throughout the body, pathogenicity is retained and arguably increased, as bacterial concentration within the biofilm increases and individuals tend to leave the biofilm, either through purposeful dissolution of EPS or through stresses on the biofilm itself by the fluid encasing the biofilm [19].

When bacteria cluster in a biofilm, movement of advantageous genetic traits, such as antibiotic resistance, throughout the constituents of the biofilm is expedited through transformation, horizontal gene transfer, or phage infection, making each individual bacterium even more virulent when it leaves the biofilm. *P. aeruginosa* biofilms, for example, exhibit a high concentration of DNA within their EPS. This also promotes significant genetic variability within a biofilm, increasing the chances that one of the many individuals will survive an environmental insult [19]. This includes antibiotics, leading to concern that excessive and inappropriate antibiotic use against biofilms expedites the development of antibiotic resistant strains [12].

### **4.3. Wound healing inhibition**

**3.4 Water and biosurfactants**

**Component Function(s)**

Polysaccharides Mechanical strength, adherence

Biosurfactants Adherence, detachment

Water Source of ions and compounds in solution

**Table 2.** Composition of biofilm exopolymeric substance (EPS) and associated functions.

Proteins Mechanical strength, adherence, detachment, virulence

eDNA Mechanical strength, adherence, antimicrobial, genetic transfer

**EPS composition**

240 Wound Healing - Current Perspectives

**4. Pathophysiology**

**4.1. Biofilm development**

**4.2. Biofilms and chronic disease**

rough [19].

Water is by far the largest component of the matrix, and water management is so critical that bacteria actively respond to desiccation by producing EPS. Molecular composition of the water component is critical as well, and the EPS matrix acts as a molecular sieve, sequestering cations, anions, nonpolar compounds and particles from the water phase. By comparison, biosurfactants have antibacterial and antifungal properties and are important for bacterial attachment and detachment from oil droplets. *Rhamnolipids*, which can act as surfactants,

Biofilms utilize a variety of mechanisms in order to establish themselves. When exposed to adverse conditions, planktonic bacteria facilitate survival by forming biofilms. This occurs through "*phase variation*" and "*adaptive mutation*," genetic alterations that include point mutations, recombination, and transpositions, with the goal of producing individuals more capable of producing biofilms. *V. cholera*, *S. typhi*, and *E. coli* all exhibit stress-induced genetic alteration by adaptive mechanisms that produce a biofilm-capable phenotype, producing distinct, wrinkled individuals. *V. cholera* produces a more chlorine-resistant subtype called rugose, while *S. typhi* and *E. coli* change to an "*rdar*" phenotype, or red, dry, and

By establishing biofilms, bacterial species not only increase their antibiotic resistance 1000-fold, they produce optimal conditions for chronic infections. By sacrificing aggressive movement throughout the body for confinement within a protective extracellular matrix, bacterial species effectively hide antigens, reduce the effectiveness of antibiotics, and blunt the immune response, promoting chronic disease: endocarditis, chronic kidney stones, and CF infections [19, 20].

have been found in the EPS matrix of *P. aeruginosa* [15] (**Table 2**).

Biofilms involve a complex relationship between bacteria virulence factors, survival mechanisms, and the host immune response [22]. Different species all exhibit particular biofilm characteristics that inhibit wound healing. EPS by itself represents a physical barrier against inflammatory cell phagocytosis, and has the potential to inhibit the complement cascade and antibiotic penetration into the wound [23]. Acellular extract from *S. aureus* biofilms inhibits the movement of keratinocytes and promotes apoptosis, leading to impaired cutaneous wound healing. This extract did not differ in pH or calcium levels; its effect on keratinocytes was due to direct cytotoxic substances secreted from or present on *S. aureus* bacterium: alphatoxin and cell surface-expressed fibronectin-binding proteins [21].

*P. aeruginosa* biofilms similarly inhibit neutrophil movement but may spare their capacity for oxidative burst, and exhibit a capacity for ejecting individual bacterium from the biofilm [24]. Another potential mechanism for *P. aeruginosa* biofilm resistance to neutrophils is the rapid necrosis induced by the production of *rhamnolipids* [23]. Additionally, significant delay in wound healing, re-epithelization and collagen deposition have been reported without significant difference in PMN infiltration or granulation tissue [6]. The ultimate result is neutrophil aggregation near the biofilm, with oxidative burst products accumulating and causing neutrophil death, while individuals within the biofilm leave to create new colonies away from the initial site [24, 25].

Biofilms in general promote a host inflammatory response that poorly penetrates the biofilm itself, causing surrounding cell damage instead [18]. Host inflammatory signal expression also characterizes the biofilm infection; in general, those with impaired host immune responses, such as those with diabetes or arterial insufficiency, tend to have more significant wounds [22].

Most literature cites the exopolymeric substance (EPS), serving as a physical barrier, as a cause of antibiotic resistance, and this is seen in some species; *P. aeruginosa* EPS contains negatively-charged alginate that easily slows the diffusion of positively-charged aminogly-

However, some specific pairs of antibiotics and species do exhibit unrestricted diffusion: ciprofloxacin and ampicillin through *K. pneumoniae*, rifampin through *S. epidermidis*, ciprofloxacin through *P. aeruginosa*, and tetracycline through *E. coli*, illustrating that although the EPS does contribute, there are many more factors, related to or independent from the EPS, that

While the EPS does indeed slow diffusion of antibiotic, eventually enough antibiotic will accumulate and kill the offending pathogen; this result has been observed in *P. aeruginosa* with tobramycin, despite the alginate produced. An important role, then, of the EPS is not blocking the antibiotic, but slowing its effect and allowing the bacteria within the biofilm to prepare. Antibiotics, for example, can stimulate the production of additional EPS in *S. epider-*

More specifically, a variety of antibiotics stimulated polysaccharide intracellular adhesion production in *S. epidermidis*, and beta-lactam antibiotics upregulated *cps* gene expression in *E. coli*, promoting the production of colonic acid; both are critical for biofilm formation in their respective species. As for *P. aeruginosa*, imipenem stimulated alginate production and the *arr*

Within the biofilm, constituent bacteria construct a hypoxic and nutrient-deprived microenvironment that slows bacterial division and, as a result, blunts the effect of antibiotics.

Alginate in mucoid *P. aeruginosa*

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 243

*S. aureus* biofilms impair keratinocytes; *P. aeruginosa* biofilms impair neutrophils

Horizontal gene transfer, eDNA, phage

*S. viridans* seeding from dental plaque to

infection, transformation

Low oxygen, nutritional state microenvironment within the biofilm

endocardium

gene was found to influence biofilm resistance to aminoglycosides [27].

**Factor Function Examples**

effect of antibiotics

and wound healing

immune system attack

Stress response genes Increased resistance to antibiotics *E. coli* rpoS gene

Genetic changes Genetic diversity, exchange of virulence

Escape behaviors Promote establishment of new colonies

Environmental alterations Reduction of bacterial division and

division

antigens, inflammatory response, and

factors and antibiotic resistance genes

away from site under antimicrobial or

susceptibility to antibiotics targeting

**Table 3.** Mechanisms by which biofilms lead to chronic disease, with associated functions and examples.

Persister phenotype Increased resistance to antibiotics *E. coli* persister genes glpD, glpABC, plsD

Establishes chronic infection and inhibits host inflammatory response

**Factors for chronic disease and antibiotic resistance in biofilms**

EPS Block host detection of bacterial

Molecular messengers/host immune modulation

cosides [22, 23, 27].

contribute in sum to resistance [27].

*midis*, *E. coli*, and *P. aeruginosa* [27].

**Figure 3.** Biofilm pathophysiology. Common pathways followed by bacteria to chronic infection and wound healing impairment.

*S. aureus* biofilms promote a distinct profile of *IL-1β* and *TNF-α* expression indicative of a mild but chronic inflammatory response [17]. While mild inflammation is helpful towards eradicating the infection by attracting an immune response and increasing collagen synthesis and granulation tissue formation, persistently high amounts of *IL-1β* and *TNF-α* decrease growth factors and increase metalloproteases, delaying resolution of the infection and wound healing [20].

*P. aeruginosa* in particular exhibits the highest virulence compared to *S. aureus* and *K. pneumoniae* due to this reason; *P. aeruginosa* biofilms exhibit the lowest bacterial counts but cause the highest elevation in *IL-1β* and *TNF-α* compared to the other two strains [23]. MRSA biofilms modulate the immune response by stimulating macrophages towards an M2 instead of M1 response, inhibiting inflammation and promoting fibrosis [26]. Chronic diseases caused by biofilms, in essence, are due to a complex equilibrium between bacterial defenses and the host immune response (**Figure 3**).

### **5. Antibiotic resistance mechanisms**

Biofilms are notoriously resistant to antibiotics, making them frustrating to treat, particularly in implanted devices, where usually the most viable solution is replacing the device entirely [27, 28]. Most literature cites the exopolymeric substance (EPS), serving as a physical barrier, as a cause of antibiotic resistance, and this is seen in some species; *P. aeruginosa* EPS contains negatively-charged alginate that easily slows the diffusion of positively-charged aminoglycosides [22, 23, 27].

However, some specific pairs of antibiotics and species do exhibit unrestricted diffusion: ciprofloxacin and ampicillin through *K. pneumoniae*, rifampin through *S. epidermidis*, ciprofloxacin through *P. aeruginosa*, and tetracycline through *E. coli*, illustrating that although the EPS does contribute, there are many more factors, related to or independent from the EPS, that contribute in sum to resistance [27].

While the EPS does indeed slow diffusion of antibiotic, eventually enough antibiotic will accumulate and kill the offending pathogen; this result has been observed in *P. aeruginosa* with tobramycin, despite the alginate produced. An important role, then, of the EPS is not blocking the antibiotic, but slowing its effect and allowing the bacteria within the biofilm to prepare. Antibiotics, for example, can stimulate the production of additional EPS in *S. epidermidis*, *E. coli*, and *P. aeruginosa* [27].

More specifically, a variety of antibiotics stimulated polysaccharide intracellular adhesion production in *S. epidermidis*, and beta-lactam antibiotics upregulated *cps* gene expression in *E. coli*, promoting the production of colonic acid; both are critical for biofilm formation in their respective species. As for *P. aeruginosa*, imipenem stimulated alginate production and the *arr* gene was found to influence biofilm resistance to aminoglycosides [27].

Within the biofilm, constituent bacteria construct a hypoxic and nutrient-deprived microenvironment that slows bacterial division and, as a result, blunts the effect of antibiotics.

*S. aureus* biofilms promote a distinct profile of *IL-1β* and *TNF-α* expression indicative of a mild but chronic inflammatory response [17]. While mild inflammation is helpful towards eradicating the infection by attracting an immune response and increasing collagen synthesis and granulation tissue formation, persistently high amounts of *IL-1β* and *TNF-α* decrease growth factors and increase metalloproteases, delaying resolution of the infection and wound healing [20].

**Figure 3.** Biofilm pathophysiology. Common pathways followed by bacteria to chronic infection and wound healing

*P. aeruginosa* in particular exhibits the highest virulence compared to *S. aureus* and *K. pneumoniae* due to this reason; *P. aeruginosa* biofilms exhibit the lowest bacterial counts but cause the highest elevation in *IL-1β* and *TNF-α* compared to the other two strains [23]. MRSA biofilms modulate the immune response by stimulating macrophages towards an M2 instead of M1 response, inhibiting inflammation and promoting fibrosis [26]. Chronic diseases caused by biofilms, in essence, are due to a complex equilibrium between bacterial defenses and the

Biofilms are notoriously resistant to antibiotics, making them frustrating to treat, particularly in implanted devices, where usually the most viable solution is replacing the device entirely [27, 28].

host immune response (**Figure 3**).

impairment.

242 Wound Healing - Current Perspectives

**5. Antibiotic resistance mechanisms**


**Table 3.** Mechanisms by which biofilms lead to chronic disease, with associated functions and examples.

For example, *E. coli* increases *cydAB* and *b2997-hybABC* genes expression. Along with this micro-environment, bacteria establish a stationary-phase state and express stress response genes; *E. coli* increases *rpoS* expression, while *P. aeruginosa* increases *groES*, *dnaK*, catalase, *katA*, and *katB* [27].

**7. Management of biofilm**

Among these, we have the following:

methicillin sensitive *S. aureus* [27].

activities except quorum-sensing [27].

bility to daptomycin [28].

Even though there is not a standard debridement type, frequent sharp and mechanical debridement have been suggested as the standard treatment for biofilm infection. Nevertheless, up to 30% [30] of biofilm infected wounds continued unresolved after these, and therefore other options are being considered, such as biological, enzymatic and autolytic [12, 30–33]. Mechanical debridement involves the application of wound dressings that expedite wound healing and resolve the biofilm infection [12]. For example, silver-based dressing is effective against *P. aeruginosa* biofilms [16]. Additionally, antimicrobial coatings, on inserted devices, for example, can hinder biofilm formation [27]. Sharp debridement, by contrast, involves scraping away at the wound with a sharp instrument to remove necrotic tissue [12]. Beyond debridement, many other treatment modalities for biofilms are being explored, including

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 245

Given the complex interactions between biofilm bacterium, the physical extracellular matrix, secreted signals and toxins, and the host immune response, there are understandably many molecular solutions for disrupting the biofilm and promoting resolution of chronic wounds.

• Furanone, a substance structurally similar to a class of quorum sensing signal produced by the marine alga *Delisea pulchra*, has been successfully used to treat *V. harvey*, *B. subtilis*, and *P. aeruginosa* biofilms. Furanone acts by disrupting quorum sensing using this similarity [27]. • Patulin, a molecule found in *Penicillium* extracts has the ability to disrupt quorum-sensing, and also was proven to be effective against *P. aeruginosa* biofilm pulmonary infection in a

• Farnesol, produced by *C. albicans* is effective against *S. aureus* biofilms by compromising its membrane integrity, additionally, it increases the effect of Gentamycin on *MRSA* and

• Ursolic acid, a natural plant extract, also disrupts *P. aeruginosa*, *V. harvey*, and *E. coli* biofilms *via* a mechanism that is not completely dilucidated, involves several bacterial metabolic

• Staphylococcal accessory regulator (*sarA*) has been identified as a key regulator for biofilm formation, and therefore is, in effect, a potential therapeutic target. sarA mutant strains of *S. aureus* and *S. epidermidis* experienced limited biofilm formation and increased suscepti-

• For MRSA in particular, due to its particular trait of promoting a fibrotic M2 response, rather than a strongly inflammatory M1 response, inserting M1 macrophages or stimulating such a response using *EP67* can prevent MRSA biofilms entirely and also resolves MRSA biofilms better than antibiotics or administration of neutrophils. *EP67* is a CD88 agonist that converts an M2 response by increasing the amount of inflammatory cytokines produced and increases the potency of macrophage movement into the biofilm [26].

molecular solutions, energy-based interventions, and new topical medications.

mouse model, acting synergistically with tobramycin [27].

Biofilm bacteria also increase the population of slow-growing "persisters," particularly hardy individual bacterium that can resist antibiotics. In *E. coli*, *glpD*, *glpABC*, *plsD*, are critically involved in persister development, as well as chromosomal toxin/antitoxin genes *relE* and *hipBA*. Finally, there are also specific biofilm-only products, such as *ndvB* in *P. aeruginosa*, that specifically target certain antibiotics, in this case tobramycin [27] (**Table 3**).

### **6. Diagnosis**

Bacterium often do not present purely in a planktonic or biofilm state; infections often contain a mixture of both. Basic criteria of the present of a biofilm are proposed by Parsek and Singh [19] and include the following: (1) bacteria are attached to a particular surface, (2) when examined, bacteria are organized into groups surrounded by EPS, (3) the infection is isolated to a particular area, and (4) the infection is difficult to treat with antibiotics despite significant eradication when in planktonic form.

Current diagnosis of wound infections is based on the bacterial side of the infection, rather than the host side; culturable CFUs is the most basic diagnostic tool but limits the diagnosis to only culturable bacteria [29]. Additionally, as biofilms are an observed mode of growth for bacteria in living hosts, it is difficult to sample a suspected host and have the bacteria establish the same biofilm on culture [19].

Furthermore, a significant amount of biofilms contain multiple species, an average of 5.4 and a maximum of 106 [18, 25]. PCR surpasses this limitation and allows clinicians to detect unculturable species, but the severity of the infection cannot be assessed in a multispecies infection [29]. There has also been success in determining biofilm formation by *P. aeruginosa* in CF patients by measuring the ratio between two quorum sensing messengers [19]. Autoinducers indicating virulence factor expression is another proposed diagnostic measurement [18].

Newer proposed tests measure the host side of the infection beyond clinical assessment, where the appearance of inflammatory signs can be unreliable and change over time. New upcoming methods of diagnosing and assessing the severity of chronic wounds revolve around measuring host inflammatory markers [29].

However, tests must be designed around each individual species' unique course and profile of inflammatory markers, as well as the unique relationship between the inflammatory marker levels and virulence; for example, *P. aeruginosa* exhibits the lowest bacterial counts but the highest *IL-1β* and *TNF-α* response, as compared to *S. aureus* and *K. pneumoniae* [22, 23].

### **7. Management of biofilm**

For example, *E. coli* increases *cydAB* and *b2997-hybABC* genes expression. Along with this micro-environment, bacteria establish a stationary-phase state and express stress response genes; *E. coli* increases *rpoS* expression, while *P. aeruginosa* increases *groES*, *dnaK*, catalase,

Biofilm bacteria also increase the population of slow-growing "persisters," particularly hardy individual bacterium that can resist antibiotics. In *E. coli*, *glpD*, *glpABC*, *plsD*, are critically involved in persister development, as well as chromosomal toxin/antitoxin genes *relE* and *hipBA*. Finally, there are also specific biofilm-only products, such as *ndvB* in *P. aeruginosa*, that

Bacterium often do not present purely in a planktonic or biofilm state; infections often contain a mixture of both. Basic criteria of the present of a biofilm are proposed by Parsek and Singh [19] and include the following: (1) bacteria are attached to a particular surface, (2) when examined, bacteria are organized into groups surrounded by EPS, (3) the infection is isolated to a particular area, and (4) the infection is difficult to treat with antibiotics despite significant

Current diagnosis of wound infections is based on the bacterial side of the infection, rather than the host side; culturable CFUs is the most basic diagnostic tool but limits the diagnosis to only culturable bacteria [29]. Additionally, as biofilms are an observed mode of growth for bacteria in living hosts, it is difficult to sample a suspected host and have the bacteria establish

Furthermore, a significant amount of biofilms contain multiple species, an average of 5.4 and a maximum of 106 [18, 25]. PCR surpasses this limitation and allows clinicians to detect unculturable species, but the severity of the infection cannot be assessed in a multispecies infection [29]. There has also been success in determining biofilm formation by *P. aeruginosa* in CF patients by measuring the ratio between two quorum sensing messengers [19]. Autoinducers indicating virulence factor expression is another proposed diagnostic

Newer proposed tests measure the host side of the infection beyond clinical assessment, where the appearance of inflammatory signs can be unreliable and change over time. New upcoming methods of diagnosing and assessing the severity of chronic wounds revolve

However, tests must be designed around each individual species' unique course and profile of inflammatory markers, as well as the unique relationship between the inflammatory marker levels and virulence; for example, *P. aeruginosa* exhibits the lowest bacterial counts but the highest *IL-1β* and *TNF-α* response, as compared to *S. aureus* and *K. pneumoniae* [22, 23].

specifically target certain antibiotics, in this case tobramycin [27] (**Table 3**).

*katA*, and *katB* [27].

244 Wound Healing - Current Perspectives

**6. Diagnosis**

eradication when in planktonic form.

the same biofilm on culture [19].

around measuring host inflammatory markers [29].

measurement [18].

Even though there is not a standard debridement type, frequent sharp and mechanical debridement have been suggested as the standard treatment for biofilm infection. Nevertheless, up to 30% [30] of biofilm infected wounds continued unresolved after these, and therefore other options are being considered, such as biological, enzymatic and autolytic [12, 30–33]. Mechanical debridement involves the application of wound dressings that expedite wound healing and resolve the biofilm infection [12]. For example, silver-based dressing is effective against *P. aeruginosa* biofilms [16]. Additionally, antimicrobial coatings, on inserted devices, for example, can hinder biofilm formation [27]. Sharp debridement, by contrast, involves scraping away at the wound with a sharp instrument to remove necrotic tissue [12]. Beyond debridement, many other treatment modalities for biofilms are being explored, including molecular solutions, energy-based interventions, and new topical medications.

Given the complex interactions between biofilm bacterium, the physical extracellular matrix, secreted signals and toxins, and the host immune response, there are understandably many molecular solutions for disrupting the biofilm and promoting resolution of chronic wounds. Among these, we have the following:


• Ribonucleic acid III inhibiting peptide (RIP) is a promising new intervention for biofilms, as it inhibits the quorum sensing necessary for biofilms to form [34]. RIP treatment accelerates wound healing in *S. aureus* and *S. epidermidis* biofilms to equal that of uninfected wounds. RIP also exhibits increased effect when combined with antibiotics in the treatment of *S. epidermidis* infections in devices [27].

Biofilms are clinically diagnosed with four basic criteria—attached, organized, local, and antibiotic resistant. Assessment with older culture methods has been proven inefficient. Modern methods such as PCR and detection of molecular inflammatory markers and secreted bacterial products are more useful methods of diagnosis. While the standard treatment is frequent and aggressive debridement, there are multiple modalities for the treatment of biofilms—biologic, enzymatic, autolytic, and mechanical—with newer molecular treatments in combination with

The Impact of Biofilm Formation on Wound Healing http://dx.doi.org/10.5772/intechopen.85020 247

The authors would like to thank our families and the Division of Plastic & Reconstructive

Division of Plastic and Reconstructive Surgery, Department of Surgery, Northwestern

[1] Sen CK.Wound healing essentials: Let there be oxygen. Wound Repair and Regeneration.

[2] Malone M, Bjarnsholt T, McBain AJ, James GA, Stoodley P, Leaper D, et al. The prevalence of biofilms in chronic wounds: A systematic review and meta-analysis of pub-

[3] Armstrong DG, Boulton AJM, Bus SA. Diabetic foot ulcers and their recurrence. The

[4] James GA, Swogger E, Wolcott R, Pulcini E, Secor P, Sestrich J, et al. Biofilms in chronic

[5] Romling U, Balsalobre C. Biofilm infections, their resilience to therapy and innovative

traditional antibiotic therapy showing promising results.

Surgery staff for their unconditional support and help.

Rafael A. Mendoza, Ji-Cheng Hsieh and Robert D. Galiano\*

lished data. Journal of Wound Care. 2017;**26**(1):20-25

New England Journal of Medicine. 2017;**376**(24):2367-2375

wounds. Wound Repair and Regeneration. 2008;**16**(1):37-44

treatment strategies. Journal of Internal Medicine. 2012;**272**(6):541-561

\*Address all correspondence to: rgaliano@nm.org

The authors declare no conflict of interest.

**Conflict of interest**

**Thanks**

**Author details**

**References**

University, Chicago, IL, USA

2009;**17**(1):1-18

• d-amino acids is a specific mix containing d-tyrosine, d-leucine, d-tryptophan and d-methionine that form a factor that was first found to prevent biofilm formation in *B. subtilis*, and later on tested on *P. aeruginosa* and *S. aureus*. In *S. aureus*, another combination (d-phenylalanine, d-proline, d-tyrosine) was found to be more effective and, more importantly, that its action is targeted to the growth stage of biofilm formation [23, 35, 36].

However, treatment cannot only consist of quorum sensing inhibitors or interventions that specifically target the biofilm, as bacteria can still survive and grow in planktonic form; daptomycin is the antibiotic of choice most effective against biofilm-forming bacteria [28].

Energy-based therapeutic options, such as ultrasound, are another viable option for treating biofilms; for *P. aeruginosa* biofilms, daily or every other day low frequency ultrasound is effective in reducing inflammation and improving wound healing [16, 37]. Additional research has also investigated the application of different topical medications on biofilm resolution and wound healing; for example, wound healing from *S. aureus* biofilms benefits from exposed desiccation or the application of honey or molasses on the wound site compared to saline, exhibiting greater granulation tissue and decreased inflammation, primarily due to the action of air or osmotic agents in drying the wound [38].

### **8. Conclusions**

Contrasting with free-floating, acutely infectious planktonic forms of bacteria, a biofilm is an aggregated colony of bacteria, usually of multiple species, that produces a protective EPS and establishes a microenvironment within that is conductive to survival and ultimately leads to chronic infection in the form of kidney stones, pulmonary infections, endocarditis, and cutaneous non-healing wounds.

When exposed to environmental stressors, bacterial undergo genetic changes that promote biofilm formation. Biofilms are made up of multiple elements—polysaccharides, proteins, extracellular DNA, and water/biosurfactants—all which have unique structural and functional traits that establishes the biofilm and its properties. Biofilms are a primary cause of chronic cutaneous wounds, due to the secretion of signals that inhibit a proper host immune response. While each species' biofilm is different in its particular properties, make-up, and response to antibiotics, biofilms are, in general, notoriously difficult to treat using antibiotics due to the EPS blocking the diffusion of antibiotics and allowing the production of a microenvironment conductive to gene transfer, metabolic slowing, selection for hardier individuals, and the development of escape behaviors that create new biofilms elsewhere in the body.

Biofilms are clinically diagnosed with four basic criteria—attached, organized, local, and antibiotic resistant. Assessment with older culture methods has been proven inefficient. Modern methods such as PCR and detection of molecular inflammatory markers and secreted bacterial products are more useful methods of diagnosis. While the standard treatment is frequent and aggressive debridement, there are multiple modalities for the treatment of biofilms—biologic, enzymatic, autolytic, and mechanical—with newer molecular treatments in combination with traditional antibiotic therapy showing promising results.

## **Conflict of interest**

The authors declare no conflict of interest.

### **Thanks**

• Ribonucleic acid III inhibiting peptide (RIP) is a promising new intervention for biofilms, as it inhibits the quorum sensing necessary for biofilms to form [34]. RIP treatment accelerates wound healing in *S. aureus* and *S. epidermidis* biofilms to equal that of uninfected wounds. RIP also exhibits increased effect when combined with antibiotics in the treatment

• d-amino acids is a specific mix containing d-tyrosine, d-leucine, d-tryptophan and d-methionine that form a factor that was first found to prevent biofilm formation in *B. subtilis*, and later on tested on *P. aeruginosa* and *S. aureus*. In *S. aureus*, another combination (d-phenylalanine, d-proline, d-tyrosine) was found to be more effective and, more importantly, that its

However, treatment cannot only consist of quorum sensing inhibitors or interventions that specifically target the biofilm, as bacteria can still survive and grow in planktonic form; daptomycin is the antibiotic of choice most effective against biofilm-forming bacteria [28].

Energy-based therapeutic options, such as ultrasound, are another viable option for treating biofilms; for *P. aeruginosa* biofilms, daily or every other day low frequency ultrasound is effective in reducing inflammation and improving wound healing [16, 37]. Additional research has also investigated the application of different topical medications on biofilm resolution and wound healing; for example, wound healing from *S. aureus* biofilms benefits from exposed desiccation or the application of honey or molasses on the wound site compared to saline, exhibiting greater granulation tissue and decreased inflammation, primarily due to the action

Contrasting with free-floating, acutely infectious planktonic forms of bacteria, a biofilm is an aggregated colony of bacteria, usually of multiple species, that produces a protective EPS and establishes a microenvironment within that is conductive to survival and ultimately leads to chronic infection in the form of kidney stones, pulmonary infections, endocarditis, and

When exposed to environmental stressors, bacterial undergo genetic changes that promote biofilm formation. Biofilms are made up of multiple elements—polysaccharides, proteins, extracellular DNA, and water/biosurfactants—all which have unique structural and functional traits that establishes the biofilm and its properties. Biofilms are a primary cause of chronic cutaneous wounds, due to the secretion of signals that inhibit a proper host immune response. While each species' biofilm is different in its particular properties, make-up, and response to antibiotics, biofilms are, in general, notoriously difficult to treat using antibiotics due to the EPS blocking the diffusion of antibiotics and allowing the production of a microenvironment conductive to gene transfer, metabolic slowing, selection for hardier individuals, and the development of escape behaviors that create new biofilms

action is targeted to the growth stage of biofilm formation [23, 35, 36].

of *S. epidermidis* infections in devices [27].

246 Wound Healing - Current Perspectives

of air or osmotic agents in drying the wound [38].

**8. Conclusions**

cutaneous non-healing wounds.

elsewhere in the body.

The authors would like to thank our families and the Division of Plastic & Reconstructive Surgery staff for their unconditional support and help.

### **Author details**

Rafael A. Mendoza, Ji-Cheng Hsieh and Robert D. Galiano\*

\*Address all correspondence to: rgaliano@nm.org

Division of Plastic and Reconstructive Surgery, Department of Surgery, Northwestern University, Chicago, IL, USA

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