tissue regeneration in loss of substance on the lower...

Tissue Regeneration in Loss of Substanceon the Lower Limbs through Use of

Platelet-Rich Plasma, Stem Cells from AdiposeTissue, and Hyaluronic Acid

Valerio Cervelli, MD; Barbara De Angelis, MD; Lucilla Lucarini, MD; Diana Spallone, MD; Alberto Balzani, MD;Ludovico Palla, MD; Pietro Gentile, MD; and Paolo Cerulli, MD

INTRODUCTIONMany plastic and reconstructive surgical procedures are performed

each year to repair soft-tissue defects that result from traumatic

injury (ie, significant burns), tumor resections (ie, mastectomy

and carcinoma removal), and congenital defects.1

The primary goal of tissue engineering is to regenerate healthy

tissues or organs for patients in need, thus eliminating the need

for tissue or organ transplants and mechanical devices.2 With

organ and tissue transplants, immunologic rejection is often a

primary concern for patients receiving donated tissues. Tissue-

engineering strategies are suggested to eliminate these concerns.

PLATELET-RICH PLASMAPlatelet-rich plasma (PRP) is developed from autologous blood

and consists of a volume of autologous plasma with a platelet

concentration above the baseline. Platelet-rich plasma has been

traditionally used as a source of platelet growth factors (GFs).

Activation of platelets induces the release of several early (basic

fibroblast GF, platelet-derived GF [PDGF], and insulin-like GF

[IGF]) and delayed GFs (epidermal GF [EGF], vascular endothe-

lialGF [VEGF], transformingGFh, and IGF). Platelets also secrete

other proteins, such as fibrinogen, vitronectin, and fibronectin,

which play a critical role in the regulation of cell–cell interactions

and cell spatial organization. Platelet GFs are able to stimulate the

replication of mesenchymal cells, such as fibroblasts, osteoblasts,

and endothelial cells, and to promote chemotaxis effect on macro-

phages, monocytes, and polymorphonuclears.

Autologous PRP (APRP) has been safely used in many disci-

plines, including orthopedics, maxillofacial surgery, cardiothoracic,

and plastic and reconstructive surgery; in particular on non-

healing wounds, such as trophic and vascular ulcers, decubitus

wounds, fistulae, burns, and dermoepidermal dystrophies. The

use of PRP has been reported as an autologous scaffold for cel-

lular growth, and it has been proven that, in association with

ADVANCES IN SKIN & WOUND CARE & VOL. 23 NO. 6 262 WWW.WOUNDCAREJOURNAL.COM

ORIGINAL INVESTIGATION

Valerio Cervelli, MD, is Director; Barbara De Angelis, MD, is a Physician; Lucilla Lucarini, MD, is a Physician; Diana Spallone, MD, is a Physician; Aberto Balzani, MD, is a Physician; Ludovico Palla, MD, is a

Physician; PietroGentile,MD, is a Physician; andPaoloCerulli, MD, is a Physician; all in theDepartment of Plastic andReconstructive Surgery, University of ‘‘Tor Vergata,’’ Rome, Italy. Acknowledgments:

None of the authors have a financial interest in any of the products, devices, or drugs mentioned in this article. Submitted March 23, 2009; accepted in revised form August 31, 2009.

ABSTRACT

BACKGROUND: Platelet-rich plasma (PRP) induces wound

regeneration and tissue repair through cell proliferation and

differentiation, promoting tissue healing and also acting as an

autologous scaffold. With a small quantity of blood, it is possible to

obtain the necessary optimal volume of PRP to treat the loss

of substance in the lower limb. It has been demonstrated that

mesenchymal stem cells are present in the adipose tissue

(thus accelerating the effect of the PRP).

METHODS: The analysis involved 30 patients with lesions

ranging from ulcerative, dystrophic, with substance loss,

with differentiating etiopathogenesis all localized on the inferior

limb, and to those treated with PRP and autologous fat grafts.

The wounds were covered with a 3-dimensional, polymerized

hyaluronic acid medicated biologic dressing. The authors’

protocol consists of a general checkup; wound examination;

instrumental, microbiological, and immunohistochemical

diagnostic examinations; and acquisition of photographic images

with follow-up at 0, 1, 2, and 3 weeks; 1, 3, and 6 months;

and 1 year.

RESULTS: The results show an improvement from minor to

moderate in 100% of patients after 3 weeks, healing in less

than 6 weeks in 47% of patients, and complete wound healing in

57% of patients within 3 months.

CONCLUSIONS: The authors’ data demonstrate the ability of the

combination of PRP and autologous adipose graft to regenerate

tissue and epithelialization with wound closure, with a significant

healing-time reduction. Furthermore, the minimally invasive

technique is well accepted by patients, with a noteworthy

improvement of the quality of life along with cost reduction

due to the fewer number of medications.

ADV SKIN WOUND CARE 2010;23:262–72

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mesenchymal stem cells,3 PRP enhances tissue regeneration.4,5

The liquid PRP and the platelet gel are obtained after centri-

fugation, from a minimal quantity of blood. When PRP is com-

bined with autologous thrombin and/or calcium chloride

platelet activation and a coagulation cascade is promoted, a

platelet gel is created.6,7 In this gelatinous substance, the

activated platelets are trapped on the fibrin network, where

they continue to excrete their contents.Meanwhile, the bioactive

substance slowly diffuses into the surrounding extracellular

environment. Platelet gel is extremely malleable. It can be

sutured to the wound bed and can be put on different vehicles,

such as advanced types of biocompatible carriers and scaffolds

(eg, fibrin or hyaluronic acid), and used topically.8 Once

prepared, the PRP can be stored for subsequent applications.

LIPOSTRUCTUREAnatomically, adipose tissue is a connective tissue derived from

the mesoderm. It contains adipoblasts, precursors of preadipo-

cytes, which further mature into adipocytes. The function of adi-

pose cells is to store triglycerides.

Fat grafts collected by liposuction can be reinjected subcuta-

neously for correction of depressed or irregular areas.9 Any anat-

omical site in which the patient has fat is acceptable as a donor

area as long as harvest does not create asymmetry. After appro-

priate centrifugation at 3000 revolutions per minute (rpm), the fat

is transferred into 1-mL syringes: the 1-mL syringes make it

technically easier to lay smaller pearls down the transplanted fat,

which should improve revascularization and survival of the fat

graft. The centrifuged fat (with Coleman technique) is composed

by a heterogenous cell population, including endothelial cells,

smooth muscle cells, pericytes, leukocytes, mast cells and multi-

potent adipose tissue–derived stem cells.10–12

The most important aspect of the technique consists in laying

down small pearls of fat with a retrograde motion. This is usually

performed on the withdrawal phase of the cannula movement,

and multiple tunnels and multiple levels are made in each area,

fanning out from the entrance sight.

The fat transfer technique has yet to be fully elucidated. There is

no standardization of the fat transfer procedure.

HYALURONIC ACIDHyaluronic acid (HA) is one of the primary extracellular matrix

(ECM) components. It is a nonsulfated, linear glycosaminoglycan

consisting of repeated units of glucuronic acid and N-acetyl-

glucosamine. Its physiological functions derive from the structural

role in ECM and its ability to interact with cell surface receptors.

The structural role is exerted throughout hygroscopic and rhe-

ological properties that allow hydration and modulation of the

cellular microenvironment. The influence on cell signaling path-

ways is ensured by cell surface receptor bindings that induce cell-

cell adhesions, cell-substrate adhesions, proliferations, and cell

migrations. It is necessary for tissue regeneration to increase the

number of cells constituting the tissue, as well as reconstruct a

structure of ECM to support the proliferation and differentiation

of cells for regeneration induction. For these reasons, it is easy to

understand the important role of HA in tissue repair processes,

facilitating the entry of a large number of cells into the injured area

and contributing to the orientation of the fibrous component of

the ECM.13,14

HA is a wound dressing that acts as a temporary dermal sub-

stitute in the treatment of wounds. It also is a 3-dimensional

scaffold that is promptly colonized by fibroblasts and ECM

components, favoring an ordered reconstruction of the dermal

tissue.15,16

MATERIALS AND METHODSDuring 2008, 30 patients (18 men, 12 women) were treated with

PRP and autologous fat grafts into the ‘‘Advanced Dressing and

Biotechnology Day Hospital’’ at the Department of Plastic and

Reconstructive Surgery, University of Rome Tor Vergata, in col-

laboration with the Transfusion Center of Policlinico Casilino,

Rome (Table 1). Eligibility criteria for the study consisted of adult

patients (age ranging from 30–87 years) affected by vascular,

diabetes-correlated, or posttraumatic diseases with ulcers or loss

of substance of the lower limbs (fingers, foot, knee, and leg).

Exclusion criteria included the presence of preexisting local and

systemic bacterial infections, renal failure (glomerularfiltration

rate <60mL/min), neoplasms, or immunohematological diseases.

The retrospective review was made by selecting patients with an

ulcer and posttraumatic injury or iatrogenic loss of substance

caused by vascular disease or diabetes correlated. The traumawas

caused by an automobile or work accident with bone or soft-

tissue exposure. Prior to enrollment, all of the wounds were

cleaned, and patients received a cycle of advanced dressing, allow-

ing for preparation of the wound bed and intraoperative surgical

debridement.

The authors’ protocol consists of a general checkup, wound ex-

amination, acquisition of photographic images, and instrumental

(echo-color Doppler), microbiological (swab culture), and immu-

nohistochemical (wound biopsy) diagnostic examinations. Pa-

tients were followed up at 0, 1, 2, and 3weeks; 1, 3, and 6months;

and 1 year. Every patientwas examined to determine the health of

the primary organs, such as the heart, liver, kidneys, and lungs

and for possible concomitant pathology. Examination of the

wound examination considered wound area (per cm3), wound

bed, wound margins, and the skin around the lesion. A swab

culture was performed to evaluate microbial infections, and the

correct systemic antibiotic therapy was administered after the

ADVANCES IN SKIN & WOUND CARE & JUNE 2010263WWW.WOUNDCAREJOURNAL.COM



antibiotic assay. To achieve a comprehensive regional site, echo-

color Doppler was conducted in the lower limbs. The Transfusion

Center assessed the suitability of the patients treated with PRP

according to its own protocol. All procedures were performed in

the operating department under local anesthesia.

The surgical steps included curettage and biopsy from the dam-

aged area (Figure 1), PRP preparation, the removal of abdominal

fat, and subsequent fat centrifugation using the Coleman

technique.

PRP PreparationA self-contained disposable kit (Regen Lab, Mollens, Switzerland)

was used to process 16 mL of venous peripheral blood. The kit

consists of 2 or more sterile evacuated blood collection tubes,

needles, and a transfer device. Blood was collected in 2 THT Regen

tubes (8mL each), and autologous thrombinwas obtained, treating

7mL of the patient’s blood using RegenATS tube. All tubes were

centrifuged at 1500 g (corresponding to 3000 rpm) using a

Hettich Rotofix Centrifuge (GMI, Inc, Ramsey, Minnesota), and

PRP was collected. The PRP obtained with a Regen Kit and a

Hettich Rotofix centrifuge, equipped with a swing-out rotor,

produces a high-quality PRP with the highest platelet recovery

and highest GF contents as published in the literature.1,2,17,18

PRP may be injected intralesionally or perilesionally or mixed

with autologous thrombin at a 9:1 ratio, forming a platelet gel

and used topically.

Fat Grafting PreparationBefore activation of the PRP, the authors infiltrated the abdomen

with 250mLofwetting solution (50mLof 1% lidocaine plus 1mL

of epinephrine 1:1000 plus 1 L of normal saline). The authors used

the Coleman microcannula technique through 2 small (3 mm)

Table 1.

PATIENT DEMOGRAPHICS AND WOUND VARIABLES

Patient Age, y Etiology Depth, mmWoundSize, cm3 Anatomical Site Comorbidity Complication

P1 80 Diabetic 1 7 � 4 Foot Cardiological disease InfectionP2 72 Vascular 0.5 10 � 5 Leg Hypertension NoP3 76 Diabetic 0.3 2 � 1.5 Fingers Cardiological disease NoP4 30 Posttraumatic 2 15 � 8 Leg No NoP5 87 Vascular 0.7 7.5 � 4 Leg and foot Neurological disease No

3 � 2P6 56 Vascular 1 12 � 9 Leg Dislipidemy NoP7 32 Posttraumatic 1.3 4.5 � 2 Ankle and foot No No

5 � 3P8 48 Posttraumatic 1.5 6.5 � 4 Ankle No NoP9 67 Diabetic 1.2 15 � 8 Foot and fingers No No

1.5 � 1P10 74 Vascular 0.5 16 � 6 Leg Hypertension NoP11 36 Posttraumatic 2 7 � 4 Ankle No NoP12 30 Posttraumatic 2.1 13 � 5 Leg and ankle No No

6 � 3P13 55 Diabetic 1 8 � 5 Foot Hypertension NoP14 85 Posttraumatic 1.7 5 � 5 Ankle Cardiological disease NoP15 60 Vascular 0.6 13 � 5 Leg No NoP16 79 Diabetic 1.3 6 � 4 Foot Renal disease InfectionP17 43 Posttraumatic 2.3 11 � 7 Knees No NoP18 39 Posttraumatic 1.7 12 � 8 Leg No NoP19 76 Diabetic 1.5 9 � 4 Ankle Renal disease NoP20 62 Vascular 0.5 5 � 6 Leg Cardiological disease NoP21 54 Posttraumatic 1 7 � 4 Leg and knees Respiratory disease NoP22 83 Diabetic 0.8 8 � 5 Leg and foot Neurological disease No

3 � 2P23 57 Posttraumatic 1 6 � 6 Foot Hypertension NoP24 78 Posttraumatic 2.3 9 � 4 Leg Renal disease NoP25 74 Vascular 1 15 � 7 Leg Respiratory disease NoP26 84 Diabetic 0.7 3 � 5 Foot and ankle Cardiological disease No

6 � 3.5P27 35 Posttraumatic 1.8 7 � 8 Leg No NoP28 46 Vascular 0.8 11 � 7 Leg Dislipidemy NoP29 41 Posttraumatic 2 12 � 4 Ankle No NoP30 87 Vascular 1 8 � 10 Foot Cardiological disease No




abdominal incisions to harvest fat tissue. Lipoaspirate was cen-

trifuged (3000 rpm for 3 minutes), obtaining fatty tissue. Refined

fat then was mixed with 0.5 mL of PRP activated and transferred

into a 1-mL Luer-Lok syringe. Next, an adipose platelet gel was

obtained and injected using the specific microcannulas.

Fat tissue was implanted at different levels in small tunnels,

previously created by forcing the cannulas (1.5-mm diameter) with

accurate and controlledmovements (Figure 2). Small quantities of

fat cells were placed in the exiting movement of the cannulas,

creating a large grid to facilitate a correct vascular development

around each fat cell. Layers of parcels of fatty tissue were posi-

tioned tomaximize the surface area of contact between the donor

and recipient tissues; separating the parcels of fat by a minuscule

amount to allow access to a blood supply enhances nutrition,

respiration, and, eventually, vascularization of the fatty tissues.

In combination with the platelet gel and fat graft application

(Figure 3), the wounds were covered with a 3-dimensional poly-

merized HA–medicated biologic dressing (Figure 4). HA is the

main component of ECM. It also is the synergistic and com-

plementary element of the PRP, having similar mechanisms of

action, allowing for the recovery and bioavailability of total GFs

platelet in biosafety. Another advantage of HA is that it can be left

in place up to 15 to 20 days,when theGFs reach their highest peak

of action, thus reducing the number and frequency ofmedications

for the benefit of the patient. In necessary cases, an elastocom-

pressionwas performed to allow a better flow at venous-lymphatic

level of the leg to interfere as little as possible with the regener-

ation process.

The healing was assessed in the following manner:

& Absent: soft-tissue exposure and absence of granulation tissue

&Minor: healing of soft-tissue exposure, presence of granulation

tissue

&Moderate: dermic and epidermic regeneration

& Complete wound healing.

Postoperative follow-up was performed with a wound exami-

nation at every week until healing was achieved; a control biopsy

at 1, 2, and 3 weeks and at 1 month; and acquisition of photo-

graphic images at 0, 1, 2, and 3weeks, at 1, 3, and 6months, and at

1 year. Atweek 1, the dressing and gauzeswere changed.Atweek

Figure 1.

POSTTRAUMATIC WOUND BEFORE TREATMENT

Figure 2.

INTRAOPERATIVE VIEW: PLATELET-RICH PLASMA AND

AUTOLOGOUS FAT GRAFT INJECTION

Figure 3.

INTRAOPERATIVE VIEW: PLATELET-RICH PLASMA GEL

APPLICATION


ADVANCES IN SKIN & WOUND CARE & JUNE 2010269WWW.WOUNDCAREJOURNAL.COMCopyright @ 20 Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.10

2, silicon was removed, and HA was applied. At week 3, HA that

was not incorporated by tissue was removed, and, if necessary,

new HA was applied until complete healing.

RESULTSThe authors’ data show an improvement fromminor tomoderate

in 100% of patients after 3 weeks, healing in less than 6 weeks

in 47% of patients, and complete wound healing in 57% of

patients within 3 months (Figure 5 and Table 3). The Kaplan-

Meier curve (Table 2) clearly showed the probability that patients

who do not achieve a complete would healing quickly, decrease

in healing time. The median of the data, corresponding to a

probability of .5, was 7.5 weeks.

Comparing the immunohistochemistry results of the intra-

operative biopsy with the postoperative biopsy (15 days after

surgery), the authors observed an increase in cell proliferation

indexes involved in wound repair (measured by proliferating cell

nuclear antigen and 5-bromodeoxyuridine for skin cells, Oil Red

O staining for adipocyte differentiation, and the VEGF for

neoangiogenesis).

The use of PRP in the authors’ patients was beneficial for

several reasons: hemostasis, promotion of healing, reduction of

the risk of material dispersion, dehiscence of fat graft, and

bacterial contamination. The authors also observed a marked

improvement, both aesthetic and functional, in the area sur-

rounding thewound, with an increase in blood supply, a restored

trophism, decreased scarring, and pain maintained over time.

The authors also evaluated the intraoperative and postoperative

adverse effects: only 2multidrug-resistant patients with diabetes

Table 2.

THE KAPLAN-MEIER CURVE

The black line is the Kaplan-Meier curve depicting the probability of patients who still do

not achieve complete wound healing. Note that the value ‘‘0’’ corresponds to the 100%

of patients with a complete healing. Gray dot lines are the confidence interval for the

Kaplan-Meier curve, and the black crosses represent the censored cases. The median of the

Kaplan-Meier curve (corresponding to a probability of .5) was at 7.5 weeks.

Figure 4.

INTRAOPERATIVE VIEW: APPLICATION OF HYALURONIC

ACID ON PLATELET-RICH PLASMA

Figure 5.

POSTOPERATIVEVIEWOFTHESAMECASEAFTER 6WEEKS




reported a partial response to the treatment with long post-

operative time (Table 1).

DISCUSSIONA wound can be further described using various parameters,

which include duration, blood flow, oxygen, hardness, inflam-

mation, pain, and coexisting systemic factors. These parameters

are clues to the definition of the cause, pathophysiology, and

status of the wound, but the authors believe that a complete and

detailed history and physical examination are also fundamental.

Wound healing is an evolutionarily conserved complex multi-

cellular process that, in skin, aims at barrier restoration. The pro-

cess involves the coordinated efforts of several cell types including

keratinocytes, fibroblasts, endothelial cells,macrophages, and plate-

lets. The migration, infiltration, proliferation, and differentiation

of these cells will culminate in an inflammatory response, the

formation of new tissue, and, ultimately, wound closure. This

complex process is executed and regulated by an equally

complex signaling network involving numerous GFs, cytokines,

and chemokines. Particular relevance is the EGF family, TGF-hfamily, fibroblast GF family, VEGF, granulocyte macrophage

colony-stimulating factor, PDGF, connective tissue GF, interleukin

family, and tumor necrosis factor a family.14 These proteins could

stimulate revascularization of the implanted adipocyte-rich gel and

constitute a 3-dimensional matrix that allows for the arrangement

of adipocytes into the correct spatial organization.19

The complex wounds are chronic injuries that tend to not heal

spontaneously within 3months. Usually, these wounds are treated

with an advanced dressing, such as biosynthetic or hydrofiber

cellulose, expanded or foam polyurethane, proteolytic enzymes,

hydrogels, hydrocolloids, negative-pressure wound therapy,

and HA.

Recently, many studies suggesting regenerative medicine pro-

cedures associated with next-generation biomaterials have been

proposed to restore an appropriate environment that encourages

healing.20–22

The objective of the authors’ study was to describe an auto-

logous new delivery system composed by APRP and autologous

fat, collected with the Coleman technique and supported by

a 3-dimensional matrix of HA for the treatment of complex

wounds. This in vivo tissue engineering preserves the

adipocyte structure and creates an optimal environment to

promote cell proliferation: endothelial cells, epidermal cells,

fibroblasts, and adipocytes. Results of numerous studies9,19,20

focusedon tissue engineering suggest 2major points for the success

of the technique: anoptimalmicroenvironment that allows correct

architectural adipocyte distribution, cell interaction, growth, and

differentiation; and an increased microcapillary network that

delivers the proper nutrient and oxygen levels to injected

cells.3,5,9,14,15 This preliminary observation established that APRP

can support an adipose tissue graft. Platelets also secrete other

proteins, such as fibrinogen, vitronectin, and fibronectin, which

play a critical role in the regulation of cell–cell interactions and

cell spatial organization. Furthermore, the use of a 3-dimensional

matrix of HA applied on the APRP mixed with adipocytes from

autologous fat improved and enhanced the tissue proliferation,

and it also has the role of containment of the autologous graft. In

this study, the authors observed that PRP and lipostructure

stimulate the regeneration and differentiation of adipose-derived

stem cell (ADSC) increase the survival of fat grafting, and retain a

3-dimensional organization. The use of HA allows the controlled

release of GF at the site of action over an extended period by

incorporating the factor into an appropriate carrier. The bio-

material behave as a physical barrier and protect transplanted

cells from immunologic attack and fibroblast infiltration.

CONCLUSIONSThis study demonstrates the role of PRP and autologous fat graft

in tissue regeneration andwound closurewith a significant healing-

time reduction. Based on the authors’ clinical practice, PRP,ADSC,

and HA are considered key elements to improve functional and

aesthetic outcomes. This association guarantees a temporary

barrier with multiple functions: reduction of pain, infection, and

wound exudate and maintenance of a moist wound environment.

In addition, the minimally invasive technique is well accepted by

patients with a noteworthy improvement of quality of life along

with cost reduction due to the fewer number of medications.

In conclusion, the results of this study show that the best treat-

ment strategy is the integration of different but complementary

disciplines, such as cell therapy, bioengineering, and biomaterial

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EVALUATION OF WOUND HEALING

Etiology Number of Patients Healing

Diabetic wounds 7 Moderate (28.5%)Complete wound healing (71.5%)

Vascular wounds 10 Complete wound healing (100%)Posttraumaticwounds

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