Wound Repair - Updates in Dressing Patents and Regeneration Biomarkers

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Recent Patents on Biomarkers 2014, 4, 133-149 133

Wound Repair - Updates in Dressing Patents and Regeneration Biomarkers

Carolina Constantin1, Georgeta Paunica-Panea

2,3, Vlad D. Constantin

2,3 and Monica Neagu

1’*

1Immunobiology Laboratory “Victor Babes” National Institute of Pathology, 99-101 Splaiul Independentei, Sect 5,

Bucharest, Postal Code 050096, Romania; 2Surgery Clinics, “Sf. Pantelimon” Emergency Hospital, Bucharest, Roma-

nia; 3“Carol Davila” University of Medicine and Pharmacy, Bucharest, Romania

Received: December 12, 2014; Accepted: January 5, 2015; Revised: January 11, 2014

Abstract: Only in the last year, over 100 reports were published focusing on innovative dressing for hard to heal wounds.

Complex technologies were developed to tackle the intricate mechanisms of wound healing and moreover to overcome

the chronic status of the wound. The essential wound management recommendations include compression and moist

wound environment maintanance. In clinical practice semi-occlusive/occlusive, antimicrobial, and advanced wound ma-

trix dressings are already implemented. In the last 10 years, several patents disclosing materials, new approaches and

technologies for biomarkers detection were published. Moreover patents that can indicate the stage of wound healing were

also disclosed. The criteria for choosing the primary dressings should be patient-oriented meeting both patient’s character-

istics and wound peculiarities, while not ignoring healthcare costs

Keywords: Biomarkers, inflammation, innovative dressings, nanotechnology, patents, proliferation, wound healing phases.

INTRODUCTION

Wound healing is a multiple process encompassing com-

plex mechanisms; the main issue in wound healing process is

the potential switch from a physiologically healing wound to

a chronic state non-healing wound. This chronic non-healing

wound is clinically demanding and the physician needs to

put in practice up-dated knowledge regarding complex mo-

lecular mechanisms and hence driving these wounds into the

normal healing cascade [1].

The wound in this case is characterized by a chronic in-

flammation that hinders the normal healing and the wound

expands its lesion(s) even months on end Fig. (1).

The wound healing process is an active and time-

dependent mechanism having three stages:

• Inflammation phase starts immediately upon wounding

and continues up to approximately 5 days. In this stage

the blood vessels contract and immediately a clot is

formed to induce haemostasis. Then the blood vessels

dilate allowing cells and important factors to access the

wounded site (white blood cells, antibodies, growth fac-

tors, enzymes and nutrients). In this stage, the known in-

flammations signs appear: erythema, heat, oedema, pain

and functional disturbance. Innate immunity cells

(neutrophils and macrophages) react immediately in this

*Address correspondence to this author at the Immunobiology Laboratory

“Victor Babes” National Institute of Pathology, 99-101 Splaiul Independen-

tei, sect 5, Bucharest, Postal Code 050096, Romania; Tel: 40 21 319 45 28; Fax: 40 21 319 45 28; E-mail: [email protected]

phase, phagocytose the microbes, dying cells and dam-

aged tissue;

• Proliferative phase can overlap a few days with the in-

flammation phase, but then it develops around 3 weeks.

In this phase, the granulation tissue appears, fibroblasts

secrete collagen, and a physical contraction is initiated

in order to reduce the tissue defect. The deposition of

collagen and extracellular matrix favours neo-

angiogenesis that nourishes fibroblasts in order to initi-

ate granulation tissue. A healthy granulation tissue has

an uneven texture and a naturally pink colour; if this tis-

sue is dark it can be an indication of reduced perfusion,

ischemia and / or infection. In the end of this phase, epi-

thelialization is induced, especially across wound’s

moist surfaces.

• Remodeling or maturation phase overlaps with the prior

phase, but it can last even 2 years; its development de-

pends on the wound type and lesion’s extension. Colla-

gen is produced to enhance the tensile strength of the

skin and thus the scar is formed. Actually, there is a re-

modeling of collagen from type III to type I. Inflamma-

tory cells are decreasing to the normal number and ac-

tivity, blood vessels regress slowly to the normal num-

ber and structure.

Wound healing is highly dependent on various intrinsic

and extrinsic factors that can drive forward a wound or can

reset phases to a chronic inflammation [2]. In chronic

wounds, this stages are completely altered and the inflamma-

.

2210-3104/14 $100.00+.00 © 2014 Bentham Science Publishers

134 Recent Patents on Biomarkers 2014, Vol. 4, No. 3 Constantin et al.

tion phase is abnormally extended, while the other phases are

insufficiently developed [3]. In chronic inflammation, the

agent that initiates the process is not eliminated, thus the

innate and adaptive immune cells are continuously triggered

enhancing the oxidative milieu. This inflammatory milieu is

sustained by reactive oxygen species, hydrolytic enzymes,

interferon- (IFN- ), various other cytokines and growth

factors. In the end, the inflammation overruns re-generation

and other deleterious processes appear: tissue destruction,

thickening and scarring of connective tissue, cells and/or

tissues death.

Chronic wounds and venous ulcers of different ethyolo-

gies can affect around 1% of Western populations. Health-

care resources can be up to a monthly cost of over 4,000$ for

an open leg ulcer. By 2016, the global market for this type

of skin pathology is estimated to a total sum of around

20 billion dollars. In Germany, it was estimated that a ve-

nous leg ulcer has an average duration of seven years, during

this time, wound dressings and nursing services costs repre-

sent the most of the health care funds [4].

The central wound management recommendations in-

clude compression and moist wound environment mainte-

nance. In clinical practice, semi-occlusive/occlusive, antimi-

crobial, and advanced wound matrix dressings are already

implemented. Clinicians agree upon the fact that choosing

the primary dressings should be patient-oriented meeting

both patient’s characteristics and wound peculiarities, while

not ignoring healthcare costs [5].

We can draw some major outlines in dressing develop-

ment, although wound care therapy and especially chronical

wounds remain an intense research area. Negative pressure

wound therapy, developed in the early 2000, involves a sub-

atmospheric pressure to the wound that is covered by a

dressing [6] and it is based on a continuous extraction of the

wound fluid increasing the local blood flow [7]. In 2011, a

novel class of products, hydroconductive dressings, was in-

troduced at the Symposium on Advanced Wound Care [1].

These new types of dressings, through a capillary technol-

ogy, draw exudates and debris from the wound. Other ex-

tremely old approach, such as hyperbaric oxygen therapy, is

still in efficacy testing phases [8]. This therapy is based on

Fig. (1). (a) Normal wound healing phases (b) and Chronic inflammation.

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Wound Dressing Patents Recent Patents on Biomarkers 2014, Vol. 4, No. 3 135

the increased oxygen level in the blood, aiding the immune

cells and the vasculatory system to proceed to wound healing

[9]. New types of biocompatible dressing that have enhanced

efficacy supported by growth factors, anti-inflammatory

molecules, antibiotics and so on, have lately gained momen-

tum in wound health care.

Recently, in wound healing domain, tissue engineering

studies thrusted especially for areas such as cosmetic surgery

and replacement of diseased tissues. The main issue in the

process of tissue repairing is the complexity of the organ:

two layers, dermis and epidermis, that harbor complex inter-

related cells and factors. Dermis is composed of connective

tissue having fibroblasts implanted in a collagen matrix. The

outer layer, epidermis has mainly keratinocytes (more than

95% of the total cell population) and important immune cells

appending to the skin’s immune specific system, specialized

dendritic cells (Langerhans cells) and melanocytes. While

the epidermis does not have vascular system, the dermis

comprises dermal capillaries [10].

Innovative technologies that use stem cells are a future

solid guarantee for skin regenerative medicine. Various stem

cells can be put in use, human embryonic stem (ES) cells,

induced pluripotent stem (iPS) cells, and mesenchymal stem

cells (MSCs) [11]. MSCs are a class of pluripotent cells,

found in various tissues, and were proven to generate all skin

cell types and moreover their use does not trigger deleterious

immune reactions [12, 13].

Although, health care market in wound management

abound, the fact that we still do not have an ideal dressing or

a perfect technology for chronical wounds resides in the lack

of cellular and molecular comprehension of the chronic

process [1]. Advanced approaches in this domain will reside

in depicting intimate characteristics not only of the wound

but as well for the patient carrying it. After establishing these

criteria the most appropriate, individualized wound care

complex approach can be chosen.

INNOVATIVE DRESSING

Innovative dressing types range from materials that ab-

sorb the exudate, to scaffolds that induce skin regeneration

and matrices that harbor cells that activate regeneration. Due

to the fact that wound healing has multiple steps where com-

plex arrays of cells and factors are involved and their activity

is orchestrated in time and space, the design of a wound

healing system or dressing, should follow some important

characteristics. Hence, an innovative dressing should have an

optimal gaseous exchange, should maintain a moist wound

environment, prevent any deleterious microbial activity and

absorb the generated exudates. An ideal dressing still does

not exist although recently we faced an explosion of publica-

tions on this topic. An array of materials are published, smart

polymeric materials, combination of various natural or syn-

thetic ones that are intended to trigger /accelerate wound

healing and moreover can deliver controlled drugs are some

of the recent published materials [14].

Dressings classification can be done in several ways:

depending on their function (debridement, antibacterial, oc-

clusive, absorbent, adherence), on the type of material (e.g.

hydrocolloid, alginate, chitosan, collagen and so on), on the

physical form (ointment, film, foam, gel), on the contact

degree with the wound (primary, secondary and island dress-

ing). Besides these classifications, probably many more cri-

teria can be brought into discussion [15]. For the sake of

highlighting the recently published patents, we have chosen

to further elaborate only on the material classification dress-

ings.

Hydrocolloid Dressings are the most widely used dress-

ings because they have properties that recommend them for a

large range of wounds. Clinically wise, they are useful be-

cause they can adhere to both moist and dry wound sites.

They are actually a family of products obtained as gel form-

ing materials (colloidal agents) that can be combined with

other materials, mainly elastomers and adhesives. The most

usual gel agent includes carboxymethylcellulose (CMC),

gelatin and/or pectin. They can be combined with other ma-

terials such as alginates.

A 3D gelatin composite hydrogel was intercalated with

soluble ciprofloxacin in a nanostructure silicate, montmoril-

lonite (MMT). In vitro drug release testing showed that there

is a controlled release up to 150h. These composite hydro-

gels induced wound healing processes in in vitro cell cul-

tures additionally maintaining their antimicrobial properties

[16].

Gelatin and poly(lactic-co-glycolic acid) (PLGA) nanofi-

bers were recently combined. The idea was to encapsulate

epidermal growth factor (EGF) in PLGA nanofibers scaf-

folds. Biocompatibility properties were tested on human fi-

broblasts investigating cell’s collagen type I and type III

gene expression. Positive results were obtained in terms of

bioactivity and hemostasis, good encapsulation capacity and

controlled release, thus another skin tissue engineering scaf-

fold and future to be wound dressing [17].

Alginate Dressings are based on calcium and sodium salts

of alginic acid. This is a polysaccharide including mannuronic

and guluronic acid units. Clinically wise, alginate dressings

have high absorbency and they will form gels upon contact

with wound exudates. These dressings can be designed as

freeze-dried porous sheets and delivered as foams or designed

as flexible fibres for large cavity wounds. Several combination

were recently published using alginate based-dressing. As

such, a new porous chitosan-alginate based polyelectrolyte

was reported. The authors demonstrate that physical proper-

ties, thickness, roughness, porosity and liquid uptake are very

good. When tested against cell lines, this new biomaterials

were not cytotoxic to L929 cell line. Authors conclude that

physicochemical properties of chitosan-alginate polyelectro-

lyte complexes, can be modified according to their clinical

utility by variation of surfactant proportion (e.g. Pluronic

F68). Hence a new biodegradable and biocompatible material

in wound dressing and/or scaffolds was reported [18].


Furthermore, in 2014, a novel gelatin-chitosan sponge

was published. The reported results showed that the material

has uniform porous structure with a high porosity, high water

uptake capacity (>1500%), and retention while the degrada-

tion percent in 28 days was in the range of 38 - 53.9%. Bio-

compatibility results proved that there is no cytotoxicity at

least for 21 days and in vivo evaluation showed that this ma-

terial favoured cell’s attachment in skin wound healing [19].

Alginates can be combined with a metal ion with antimi-

crobial activity. Alginate combined with copper showed

antibacterial activity against Escherichia coli, Staphylococcus

aureus, methicillin-resistant Staphylococcus aureus (MRSA),

Staphylococcus epidermidis and Streptococcus pyogenes. The

antibacterial activity of the combined material was propor-

tional to the Cu(2+

) ion concentration [20].

Alginates can incorporate drugs, like gentamicin sulphate

(GS) and can be formatted into alginate/pectin nanoparticles.

This nanoparticles had a good moisture transmission avoid-

ing hence wound dehydration or occlusion. The encapsulated

GS, tested in cell lines had a total permeation time of 3-6

days. Antimicrobial tests performed against Staphylococcus

aureus and Pseudomonas aeruginosa showed prolonged

antimicrobial effect of the nanoparticles when compared to

GS alone in both short and long time administration (up to

12 days) [21].

Sodium alginate (SA)/poly(vinyl alcohol) (PVA)/moxifl-

oxacin hydrochloride (MH) nanofibrous membranes (NFM)

combination were recently reported. This alginate-based

dressing had antibacterial effect against Pseudomonas aeru-

ginosa and Staphylococcus aureus. Testing performed in

animal models showed that 80% of the antibiotic MH was

released after 10 h. In vitro and in vivo results suggested for

this MH/PVA/SA nanofibers dressing future clinical applica-

tions [22].

Complex gels were designed from fully interpenetrating

networks (IPNs) using collagen-I and alginate. The idea be-

hind the designed gel is to develop a matrix with physiologi-

cal mechanical properties matching the biology of resident

cells in order to repair and regenerate the skin. Dermal fibro-

blasts cultures were encapsulated in this 3D matrix. When

different combinations of collagen and alginate were per-

formed, different cell morphologies were obtained. An en-

hanced stiffness of the matrix induced an up-regulation of

inflammation mediators (e.g. IL-10 and COX-2). Interesting

results are highlighted by the authors showing that matrix

mechanical properties can regulate the phases of wound

healing [23].

Chitosan and alginates are treated in the same category of

dressings. Chitosan and chitin based nanostructured materi-

als ‘flooded’ the recent published literature. Nanofibers from

chitin and chitosan have good biological properties in terms

of biodegradability, biocompatibility, antibacterial activity,

low immunogenicity and increased wound healing capacity.

Moreover, they can be used as drug delivery systems and

tissue engineering scaffolds [24]. Recently, hydrophobic

chitosan sponges were developed for further application as

drug-sustained-release, porous wound dressing [25]. Chito-

san (CTS) nanofibers with various fractions of silver

nanoparticles (AgNPs) were recently obtained. These nan-

ofibers have antibacterial activity (gram-negative Pseudo-

monas aeruginosa, and gram-positive methicillin-resistant

Staphylococcus aureus), thus bases for effective future dress-

ing in infected wounds [26]. Combination of chitosan with

agarose resulted in a hydrogel with bactericidal activity. In

experimental wounds, the lack of a reactive or a granuloma-

tous inflammatory reaction demonstrated its near future ap-

plication as a wound dressing [27]. Chitosan incorporated

with lysostaphin, an enzyme with bactericidal activity

against Staphylococcus genus, was confirmed as good anti-

bacterial activity in human and bovine skin infections [28].

Materials incorporating chitosan/sericin nanofibers were

proven as well biocompatible, promoting cell proliferation,

while hindering Gram-positive and Gram-negative bacteria

functions [29].

Biomaterials containing regenerated cellulose (RC) and

chitosan (Ch) combined with silver nanoparticles (AgNP)

and antibiotic gentamicin (G) were also reported. In animal

models, increased healing was obtained with RC-Ch-Ag and

RC-Ch-Ag-G combinations thus base for new wound dress-

ing material to be applied in humans [30].

In 2014, an interesting publication reported the use of

chitosan hydrogel for the detection of enzymes, as an infec-

tion-sensing wound dressing. Chitosan is functionalized with

a fluorogenic substrate, e.g alanyl-alanyl-phenylalanine-7-

amido-4-methylcoumarin (AAP-AMC) for the detection of

an active serine protease -chymotrypsin. After optimization,

this new hydrogel can indicate the presence of -

chymotrypsin in less than 5 min at a concentration as low as

10 nM. This sensing dressing could be adapted to specify a

potential infection of the wound [31].

Hydrogel Dressings. In wound care, hydrogel dressings

are designed to retain moisture on the wound’s surface and

hence aid immune system’s cells to eliminate damaged tissue

and start the healing proccess. Using these dressings pa-

tient’s pain is reduced and on the wound level it can provide

an anti-infectious barrier for skin. These dressings have

around 90% water, keeping the tissue well hydrated, but they

do not absorb the wound’s exudate. For these reasons, they

are not used per se in low drainage or infected wounds, un-

less they are actually combined with other types of dressings.

Hydrogel dressings are made out of synthetic polymers such

as poly(methacrylates) and polyvinylpyrrolidine (PVP).

These poly.mers are not inherently bioactive, thus PVP can

be blended with salicylic acid (SA)-based poly(anhydride-

esters) (SAPAE). This materials exhibit hydrogel properties.

In vitro studies published in 2014, demonstrated that SA was

released over 3-4 days and that the polymer blends signifi-

cantly the inflammatory cytokine, TNF- , without negative

effects [32].


Fibers PVP-indomethacin (INDO) were developed in

order to design a controlled release system. The complete

drug release was in 45 minutes, thus a low cost fibers having

active agent / drug rapid release properties was reported [33].

A novel composite hydrogel was recently reported com-

prising poly (vinyl alcohol) (PVA) with lysine (Lys) and

vanillin (V). This composed hydrogel PVA/Lys/V has good

antibacterial activity (against gram-negative Escherichia coli

and gram-positive Staphylococcus aureus) and in in vivo

experimental wounds this material displayed good healing

results. Moreover, after one week, the wound burns dressed

with this hydrogel had already regenerating epidermis, with

capillary new vessels. These recent combined hydrogel

PVA/Lys/V is reported as effective especially in burns without

eliminating their utilization in other types of skin wounds [34].

Polymers in Wound Healing

Polymeric biomaterials were first developed for severe

hemorrhage control, recently their potential was broaden to

wound dressing agents related to the condition / type of the

wound (acute, chronic, superficial, and full thickness) and to

the phases of the wound healing process. The use of bio-

polymers depends on their biocompatibility, biodegradabil-

ity, non-immunogenicity and mechanical properties [35].

Poly(lactide-co-glycolic acid) scaffolds alone or in com-

bination are subject of intense research. Hybrid membranes

with silk fibroin (SF) and poly(lactide-co-glycolic acid)

(PLGA) have been reported as having increased attachment

and proliferation capacity in in vitro cell cultures. In vivo

experimental models showed that residual wound area

treated with the new hybrid dressing was significantly

smaller in comparison to controls [36].

New polymers were designed with negatively charged 3-

sulfopropyl methacrylate (SA) and positively charged [2-

(methacryloyloxy)ethyl] trimethylammonium (TMA) onto

expanded polytetrafluoroethylene (ePTFE) membranes.

These polymers were assesed for hydration property, resis-

tance to fibrinogen adsorption, hemocompatibility, resistance

to fibroblast attachment and bacteria colonization. In mouse

model, using this polymer, complete re-epithelialization was

observed and new tissues were generated after 14 days.

Authors are optimistic and highlighted that these mixed-

charge copolymers are the newest generation of biomaterials

for wound dressings [37].

Another recent report shows complex nanofibers made of

poly(D, L-lactide) (PDLLA) and poly(ethylene oxide) where

2,3-dihydroxybenzoic acid (DHBA) is incorporated. DHBA

was released in 2h from the nanofibers and inhibited the

growth of several bacteria strains (Pseudomonas aeruginosa,

Klebsiella pneumoniae, Escherichia coli, Salmonella typhi-

murium and Staphylococcus aureus). Probably, the rapid

diffusion of DHBA from the nanofibers was based on the

hydrogen bonds that DHBA established with the C=O

groups from PDLLA, bonds that increased the thermal sta-

bility of the nanofiber mesh [38].

Citrate-based polymers are highly biocompatible proving

also antimicrobial activity. Studying different polymers

types, poly-octamethylene citrate had the best antimicrobial

effect. It is interesting that the intrinsic antibacterial proper-

ties in citrate-based polymers enable them to inhibit bacteria

without antibiotics/silver nanoparticles, or other anti-

bacterial compounds. Thus, citrate-based polymers have a

good medical potential once more when antimicrobial action

is intended [39].

Bio-nanotextiles, an emerging domain with an evolving

technology, can be the source of new wound dressings and

new tissue scaffolds. Silk fibroin (SF) has good biocompati-

bility, permeability, biodegradability, morphologic flexibil-

ity, and proper mechanical properties. Antibacterial polyeth-

ylenimine (PEI) was introduced in SF and, besides cytotox-

icity evaluations on L929 fibroblasts, this new bio-

nanotextiles proved a strong antibacterial activity against

Staphylococcus aureus and Pseudomonas aeruginosa [40].

Chronic wounds have triggered the development of anti-

biofilm components that make the biofilm-embedded bacte-

ria sensitive to antibiotics. An antibiofilm enzyme-based

wound spray was developed combined with an antimicrobial

compound for treating chronic wounds. In experimental

mouse models, this combination performed very well in me-

thicillin-resistant Staphylococcus aureus experimental infec-

tion. The recently published in vivo model will be further

developed in future clinical trials [41].

Several metal-nanostructures were developed, such as

gold-tellurium nanostructures (Au-Te NSs), silver-tellurium

nanostructures (Ag-Te NSs), and gold/silver-tellurium

nanostructures (Au/Ag-Te NSs). This nanostructures were

developed due to their antimicrobial activity against several

strains such as Escherichia coli, Staphylococcus enteridis

and Staphylococcus aureus. The best antibacterial activity

was proven for Au/Ag-Te NSs due to a double action: Ag(+)

ions release and Te-related ions that can generate ROS with

deleterious effects on bacteria. Authors show that these

nanostructures can be inserted in wound dressing, proving

also good biocompatibility and low-cost fabrication [42].

Silver, as indicated above, is increasingly used in wound

dressings formulations. The main purpose is to control bacte-

ria infecting wounds. In vivo bacteria are likely to exist in

biofilms, this status is clinically challenging as both control

and eradication. Two agents (ethylenediaminetetraacetic acid

(EDTA) and benzethonium chloride (BC)) designed to dis-

rupt biofilms, were incorporated to an already approved

wound dressing and supplemented with silver (AAg + E). In

the presence of AAg + E, the biofilm was eradicated through

a synergistic action of EDTA and BC while silver had per-

formed its bactericidal activity [43].


Comparing Efficacy

In search of the best wound dressing, recently, an exten-

sive comparison was published for the commercially avail-

able dressings. The most commonly five used materials were

tested on pig’s skin. Hence, Xeroform (fine mesh gauze

impregnated with a blend of 3% bismuth tribromophenate),

Opsite (polyurethane film), Kaltostat (calcium sodium algi-

nate), DuoDERM (hydrocolloid), Aquacel (hydrofiber), and

Mepilex (silicone foam) were tested. Out of all, the hydro-

colloid dressing elicited the greatest re-epithelialization

property (over 80%) while the silicone foam the lowest (over

30%). After 5 days, all dressings exhibited complete re-

epithelialization except the silicone foam. Neither infections,

nor inflammation was registered in all the tests. The silicone

foam was the easiest to use, whereas the hydrofiber, calcium

sodium alginate and polyurethane film were the most diffi-

cult to handle. After thorough evaluation, the gauze and the

hydrocolloid proved to be overall the most effective type of

dressings. While the hydrocolloid has the best re-

epithelialization, the gauze was the least expensive, easy to

use, and demonstrated rapid re-epithelialization. The evalua-

tion report recommended that the gauze should be used for

large areas, while the hydrocolloid for smaller, difficult to

treat areas [44].

Cells and Factors for Skin Regeneration

Besides the wound dressing types described above, there

are recent publications focusing on cells and specific factors

that initiate and further aid skin regeneration phases. In ani-

mal model, a surgically induced dermal wound in rat tested

the healing capacity of a novel gel composed of chitosan,

dextran sulfate and polyvinylpyrrolidone K30 (CDP). CDP

gels embeded with 20μg/mL EGF would induce in vivo a

superior wound healing process. This dressing significantly

reduced the wound defect and enhanced epithelization in

comparison to CDP gel without the growth factor [45]. In

humans, a gel containing beta urogastrone (rhEGF) was

tested in diabetic foot ulcers against the patients treated with

classical betadine dressing. The follow-up was done for 2-8

weeks and the results showed that the gel containing the

growth factor significantly reduced wound healing time with

an improved wound closure [46]. In venous leg ulcers, colla-

gen-gel matrix containing EGF was tested in patients that

were followed-up for 1-3 months. The average wound sur-

face was significantly reduced, the dressing was evaluated as

very easy and generally well tolerated, thus another good

clinical option for leg ulcers [47].

Other growth factors, such as recombinant human granulo-

cyte-macrophage colony-stimulating factor (rhGM-CSF) incor-

porated in alginate was tested in patients with chronic skin ul-

cers. Compared to the control group, an enhanced granulation

tissue, better re-epithelialization and reduced wound pain were

obtained in patients receiving rhGM-CSF [48].

Cells, as direct wound healing players, were in the last

years the main subject of research in skin regeneration do-

main. In animal model, the combination of dermal matrix

with autologous / allogenic cells reduced the inflammation

phase and accelerated the initiation of granulation tissue.

Using mesenchymal multipotent stromal cells, an important

medical need can be met for extensive wounds resulted from

important physical injuries [49].

Genetically-modified hair follicle stem cells (HFSCs)

were introduced in 3D Gel-C6S-HA (gelatin-chondroitin-6-

sulfate-hyaluronic acid) scaffolds and tested in animal mod-

els. Electron transmission microscopy showed that cells were

adhering and growing on the scaffold. The results showed

that HFSCs genetically modified for VEGF, actively pro-

moted angiogenesis and intensively stimulated wound heal-

ing [50]. Also a hyaluronic acid-based therapy tested in dia-

betic wound patients showed that autologous fibroblasts can

induce complete ulcer healing in over 80% of the patients,

compared to the 30% healing in controls. The healing time

was also reduced to around 35 days in comparison to the 48

days in controls. The combination of autologous fibroblast-

hyaluronic acid offers a new treatment in diabetic-related

wounds [51].

PATENTS UNDERLYING WOUND HEALING - MA-

TERIALS AND HEALING BIOMARKERS

There is an array of patents in this field spanning from

devices that diagnose the healing process, biocompatible

synthetic polymers or natural ones, through scaffolds that

harbor cells and/or factors that induce skin regeneration.

Thus, in 2012, a patent claimed a device that indicates

the wound’s healing phase based on the detection of at least

one biomarker [52]. With this device the physician can de-

termine if a wound is healing or if the healing is delayed.

The wound dressing can be a polyurethane film with incor-

porated detection compounds like aptamers or antibodies

that detect the presence of one or more biomarkers from two

groups (see Table 1). The patent detects, for example the

first biomarker as MPO (group 1) and the second biomarker

FGF-2 (group 2).

The phases of wound healing are evaluated by detecting

and further comparing in the wound sample the amount of a

first biomarker and the amount of a second biomarker. The

relation between the amount of the first biomarker to the

amount of the second one indicates the wound healing phase.

The analyzed sample can be a wound exudate, an aspirated

fluid, a tissue sample extracted from the dressing material

(upon removal of the dressing from the wound) or from non-

necrotic tissue removed during debridement.

If the first biomarker (group 1) is secreted by neutrophils

and the second biomarker (group 2) is secreted by fibro-

blasts, and if the amount of the first one is higher compared

to the second one, then the inflammatory phase is indicated.

If the case is opposite than the proliferative phase is indi-

cated. Regarding the actual difference in the detected amount


of biomarkers indicating one phase or the other, authors

claim a significant doubling of the amount between group 1

and group 2 of biomarkers.

The actual dressings that can be used for detection of the

biomarkers comprise an array of coverings or support matri-

ces as described in Table 2.

As detection methods for biomarkers any type of classi-

cal method can be used (ELISA, immunofluorescence test,

microarray, luminescence test, radioimmunoassay, Western

blot or dot blot). If the levels are expected to be very low,

additional technology can be used such as mass spectrome-

try, Fourier transform infrared spectroscopy (FTIR), polym-

erase chain reaction (PCR), quantitative real-time PCR, or

Northern blot.

A diagram depicting the major steps in this type of patent

is presented in Fig. (2).

Another patent claimed in 2013 reports a sampling de-

vice for wounds and it comprises a biodegradable porous

scaffold that is put in contact with the sampled tissue [53].

The scaffold contains a reversibly thermo-switchable gel.

This sampling device is a diagnostic method that can be eas-

ily applied to a variety of wounds, monitoring the physio-

logical status of associated soft tissue and depicting any un-

derlying pathologies. Moreover, this device can be used for a

particular therapy delivery to the wound. It can be used for

detection of various biomarkers, for detection of microbial

product(s)/cell(s), for a substance associated with the im-

mune response to the infection and/or for detection of sub-

stances resulted from an endocrine or metabolic condition

(e.g. diabetes, hypoxia, sepsis, biofilms or fibrosis).

Like in the previous patent [52], in this one [53], detec-

tion of a marker is claimed, indicating the healing process

phase and/or how far into the healing phase the wound is.

Moreover, the patent can indicate the level when an infection

or other pathological condition might develop. Thus, the

early diagnosis can point toward a therapy for microbe eradi-

cation, creating hence a favourable tissue environment for

tissue repair. Conventional wound sampling devices are dif-

ficult to apply in case of acute and chronic wounds, thus this

new sampling method applied in wound care would offer

physicians, plastic surgeons, dermatologists, a sampling de-

vice combined with a diagnostic assay. If there is an anti-

microbial treatment, the marker would identify the level of

infection, hence therapy efficacy. The markers that can be

monitored in this sampling device are described in Table 3

and can comprise microbial product and/or microbial cells.

This patent [53] claims a biodegradable porous scaffold

that contains a fluid to be put in contact with the tissue that

will be sampled. The fluid within the scaffold provides a

medium in which components from the sampled tissue can

be infiltrated. Then, in a conventional point-of-care test

(POCT) or point-of-use diagnostic assay the extracted fluid

can be analyzed. The scaffold can comprise as fluid various

media, from mere water to hydrogels or thermo-switchable

gel materials. These later ones can switch from liquid to gel

at a given temperature or over a given temperature range.

These materials can be polymers and copolymers based on

polylactide, poly(lactide-co-glycolide), polycaprolactone,

poly(propylene oxide), poly(butylene oxide), polyvinyl

methyl ether), poly(/V-isopropylacrylamide), poly[2-(/V-

morpholino)ethyl methacrylate], and poly[2-(dimethylamino)

ethyl methacrylate], and derivatives of these materials. The

claimed mechanism shows that, this gel soakes into the

wound fluids, hence absorbs the wound markers and then

upon analysis can be turned into liquid for sample handling

or stored at 10-15°C to reverse the gelation. The patent can

be used in two modalities: the scaffold may be loaded with

the fluid and then placed on the wound, or the scaffold may

be placed in a wound and then immediately loaded with the

fluid or at various time points. Several applications of the

fluid can be done to the same scaffold and the fluid may

penetrate entirely or only partially into the scaffold. Moreo-

ver, the scaffold can be seeded with regenerative tissue cells

if the physician indicates. In this case markers that

Table 1. Biomarkers Used Individually or in Combination for Identifying the Healing Grade of a Wound - Patent [52].

Group Biomarker Cells that Secrete the Biomarker

1 Myeloperoxidase (MPO), neutrophil elastase (nElastase), Human Neutrophil Lipolin (HNL), Lac-

toferrin, Lysozyme, Neutrophil Gelatinase-Associated Lipocalin (NGAL), Human Neutrophil Elas-

tase Anti-Neutrophil Cytoplasmic Antibodies (HNE ANCA's), MMP9, Proteinase 3, Serpin Peptidase

Inhibitor Clade B and D, Reactive Oxygen Species (ROS), or Reactive Nitrogen Species (RNS)

Neutrophil

Basic fibroblast growth factor (FGF-2), Fibroblast Growth Factor-10 (FGF-10), Fibroblast-specific

protein 1 (FSP1), prolyl-4-hydroxylase (5B5), Insulin Growth Factor-1 (IGF-1), Tetranectin, Colla-

gen alpha 1, 2, and 3 chains, SERPINA1, or Complement Components

Fibroblast 2

Calgranulin A/B, Cystatin A, S100 Calcium Binding Proteins, CD163, CD204, CD206, AM-3K,

CSF-1R (colony-stimulating factor-1 receptor), a specific marker of macrophages, EMR1 (epidermal

growth factor module-containing mucin-like receptor 1), F4/80, pro-collagen, collagen, or fibronectin

M1 macrophages initiating an

inflammatory response

M2 macrophages initiating repair

and angiogenesis


Table 2. Wound Dressing Types that Can Be Subjected to Detection of Healing Biomarkers [52].

Dressing type Components

Films (semipermeable or a semi-occlusive) Polyurethane copolymers, polyurethane film, acrylamides, acrylates, paraf-

fin, polysaccharides, cellophane and lanolin

Hydrocolloids (flexible foam, formulated in polyurethane, or formulated as

an adhesive mass such as polyisobutylene)

Carboxymethylcellulose protein constituents of gelatin, pectin, and complex

polysaccharides including Acacia gum, guar gum and karaya

Polymers (80% - 90% water) conventionally formulated as sheets, powders,

pastes and gels in conjunction with cross-linked polymers such as polyeth-

ylene oxide, polyvinyl pyrollidone, acrylamide, propylene glycol

Agar, starch or propylene glycol

Foams (hydrophilic open-celled contact surface and hydrophobic closed-cell

polyurethane)

Polysaccharide

Impregnates Pine mesh gauze, paraffin and lanolin-coated gauze, polyethylene glycol-

coated gauze, knitted viscose, rayon, and polyester

Cellulose-like polysaccharide formulated as non-woven composites of fi-

bers or spun into woven composites

Alginates, including calcium alginate

Fig. (2). Diagram of biomarker evaluation stages for wound healing. In the dressing, specific antibodies can be embedded. These antibodies

would detect from the biological sample the presence of the specific biomarkers (wound biomarker detection). Further, these biomarkers are

evaluated as concentration and cellular origin (biomarker evaluation). If molecules secreted by inflammatory cells like neutrophils prevail

upon those secreted by fibroblasts the inflammatory phase of wound healing is detected and vice versa, if fibroblast-related molecules are

more abundant the proliferation phase is indicated. Upon this evaluation, the therapy can be monitored and/or adapted to the detected phases (wound healing phase evaluation).

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Table 3. Microbial-Related and Immune-Response-Related Biomarkers that Can Be Detected for Monitoring Wound Infection

[53].

Enzymes: oxidase, lipase, tryptophanase, beta-lactamase, beta-lactamase inhibitor, esterase, dehydrogenase, kinase, hydrolase,

protease, nuclease, phosphatase, decarboxylase, and/or carboxylase

Metabolic-related factors: adenosine triphosphate (ATP), a pyridine nucleotide such as nicotinamide adenine dinucleotide (NADH)

or a flavin such as flavin adenine dinucleotide (FADH)

Exotoxin (superantigen), enterotoxin, pore - forming toxin

Microbial product

Bacterial autoinducer signaling molecules: homoserine lactone derivatives, oxo-alkyl derivatives, autoinducer-2, and competence

stimulating peptides

Microbial cells Staphylococcus epidermidis, Staphylococcus aureus, Corynebacterium, Brevibacterium, Proprionibacterium acnes, Pityrosporum,

Candida albicans; and microorganisms such as coagulase-negative Streptococci, E. Coli, Proteus, Klebsiella pneumonia or

anaerobes like Pseudomonas aeruginosa, Acinetobacter, Stenotrophomonas maltophilia

Immune response Enzymes: lysozyme, complement and phospholipase A2, antimicrobial peptides such as cathelicidin and the -defensins, and

immunoglobulin A.

Markers for inflammatory phase, or early stage expression markers (up to day 2): PDGF, TGF- , KRT17, K6HF, TNF- , IGF-1,

CXCR4, CD68, IL1 , IL10, IFNv, CD44 and CD1 1 C

Oxidative stress markers: iron II and iron III salts

Proteases: serine proteases, like elastase, thrombin; cysteine proteases matrix metalloproteases, like collagenase; and carboxyl (acid)

proteases; and endotoxins and/or inflammatories, such as lipopolysaccharides, and histamine.

Markers for proliferative phase or late expression markers (days 4-8): cell migration and proliferation factors, such as VEGF, BFGF,

IL10, MMP2, CTGF, MMP-9, IL2, IL4, IL6, IL8, CD99, type V collagen, FGF, VEGFR, NRP-1, Ang1 and Ang2, PDGFR, MCP-1,

3, 5 and 5 , VE-cadherin, CD31, ephrin, plasminogen activators, eNOS, COX-2 AC133 and Id1/ld3

Extracellular matrix proteins: collagen, hyaluronic acid, fibronectin, laminin, elastin, proteoglycans such as agrin, perlecan, versican,

decorin and fibromodulin, and sulphated glycosaminoglycans (GAGs) (e.g. heparin sulphate, chondroitin sulphate and keratin sulphate

Continuous expression markers: HSP70, MIF, CD6, TIMP1 and TIMP2

Reduced expression markers: PIP5K28, endothelial-1, TYRP1 and KRT2A

Cells characteristic for proliferative phase: fibroblasts, keratinocytes, endothelial cells, inflammatory cells.

Markers for an endocrine or metabolic condition: islet cell cytoplasmic autoantibodies, glutamic acid decarboxylase antibodies,

insulinoma-associated-2 autoantibodies, C-peptide, insulin autoantibodies, enzyme (e.g. -glucuronidase, N-acetyl- -

glucosaminidase, acid phosphatase, amylase, alkaline phosphatase, trehalase, aldolase, arginase, lipase, cholinesterase).

Markers for cell proliferation and migration: VEGF, BFGF, IL10, MMP2, CTGF, MMP-9, IL2, IL4, IL6, IL8, CD99, type V

collagen, FGF, VEGFR, NRP-1, Ang1 and Ang2, PDGFR, MCP-1, 3, 5 and 5 , VE-cadherin, CD31, ephrin, plasminogen

activators, eNOS, COX-2 AC133 Id1/ld3

should be identified are more difficult because there are new

added cells to the wound. During regeneration, these new

cells can express different markers. When the scaffold is

used as a sampling device, and wound fluid needs to be col-

lected, the contact time between the scaffold and wound can

be from 1 minute to 48 hr.

When used as a therapy device, it can incorporate a

therapeutical agent, an antimicrobial compound (e.g. silver,

iodine or chlorhexidine), an agent that improves scar resolu-

tion and/or prevents scar formation (e.g. insulin, vitamin B,

hyaluronic acid, mitomycin C, growth factors TGFbeta, cy-

tokines, corticosteroids) and/or agents that promote re-

epithelialisation.

Another patent [54] seeks to provide an intelligent dressing

system for the treatment of leg ulcers. This dressing system

enables information collection from the wound through incor-

porated sensor technologies (temperature, pH, moisture and

cell impedance). These sensors can be monitored within a

hospital or even at home and transmitted to the health care

professional through telecommunications. This dressing is

capable to induce regeneration through electromagnetic stimu-

lation because it is equipped with an electromagnetic coil,

creating a pulsed electromagnetic wave. This incorporated coil

could also support the release of ions in solution from a reser-

voir introduced in the dressing. In fact, the dressing comprises

a sensor array printed on a flexible substrate that measures at

least two or more parameters: temperature, moisture, imped-

ance, pH, particular compounds or gases, light absorption or

light transmission. These sensors are arranged in a two dimen-

sional triangulation offering the best clinical decisions upon

wound state.


This dressing for intelligent wound management is com-

prised out of several layers:

• the first one is based on nano-fibre technology and it is

in contact with the wound, absorbing and holding exu-

dates, it can also withhold drugs, e.g. a specific antibi-

otic;

• second layer can comprise a transparent material, made

as an indicative membrane for the presence of particular

exudate matter and an early warning for physician or pa-

tient;

• third encapsulation layer can support sensors for the

measurement of cell impedance, pH value and other

compounds levels; the electrical connections are de-

signed to be hosted by two additional layers.

Another set of patents [55-57] claimed by a similar group

of researchers is the enhancement of wound regeneration

through gap junctions restoration. Gap junctions facilitate

direct cell-cell communication [58] and are involved in de-

layed-healing wounds, incompletely healing wounds, and

chronic wounds. The patent claims the use of one or more

connexin inhibitors with anti-connexin peptides, gap junc-

tion closing compounds, hemichannel closing compounds,

and connexin carboxy-terminal polypeptides.

The therapeutical approach comprising one or more anti-

connexin polynucleotides can be administered within 24h

and can be used for various chronic wounds having as

pathoetiology (e.g. diabetic ulcer, diabetic foot ulcer, venous

ulcer, venous stasis ulcer, pressure ulcer, decubitus ulcer,

vasculitic ulcer, arterial ulcer, infectious ulcer, burn ulcer,

trauma-induced ulcer, ulceration associated with pyoderma

gangrenosum). The combination can be selected as shown in

Table 4 from a primary group of anti-connexin agents and

the second one selected to subtract the first type of anti-

connexin agents.

The patent claims a formulation for topical delivery as a

foam, spray or gel. The gel can be a polyoxyethylene-

polyoxypropylene copolymer-based gel or a carboxymethyl

cellulose-based gel or a pluronic gel. The recommended

doses are in the range of 0.01 - 0.050 mg/kg body weight.

Doses may be applied 3-7 days apart, or more. In the case of

a chronic wound, repeated applications can be made (e.g.

weekly, bi-weekly, monthly or other frequency) when the

physician recommends it [55].

Another patent related to wound healing is the admini-

stration of a complement inhibitor to inhibit complement

activation, particularly through C3, C5 or C5a signaling [59].

The cellular process this patent is based on, is that the acti-

vated complement system is an essential effector mechanism

of the innate immune as a rapid immune response to tissue

injury. Out of the entire complement cascade, C3a and C5a

are the most potent chemoattractants upon activation of the

complement system [60]. C3a and C5a attract neutrophils,

monocytes, and macrophages to the sites where complement

cascade was activated [61]. Activated immunity cells, like

macrophages, can also produce C3 and activate phagocytosis

and/or clearance of apoptotic and necrotic cells [62]. Moreo-

ver, C5a receptor (C5aR or CD88) signaling in Toll-like re-

ceptor (TLR)-activated macrophages selectively inhibits the

transcription of genes that encode the IL-12 cytokine family,

which in turn drives the polarization and recruitment of T-

helper lymphocytes 1 (Thl) [63]. While complement activa-

tion is needed to restore tissue injury, inappropriate comple-

ment activation can cause injury and contribute to further

tissue damage [64]. In this light, the patent [59], claims that

for chronic wounds, administering therapeutically effective

complement inhibitors, that can control complement activa-

tion, a healing process prevails. Thus, one or more inhibitors

for C3, C3aR, C5a, C5aR, factor D, factor B, C4, Clq, or any

combination of these inhibitors would be efficient (Table 5).

The patent foresees a systemic administration or a lo-

cally/topically administration of these inhibitors. This ad-

ministration can be done together or sequentially with other

wound therapies and this administration can continue until

the chronic wound heals. The patent is based on the results

obtained in animal models. Genetically deficient animals for

C3, C5 and the C5a receptor have accelerated healing of

cutaneous wounds, in comparison to their wild-type counter-

parts.

When topical administration is foreseen, besides classical

dressing, the patent recommends an enhancer that induces an

increased penetration of the therapeutical agent such as al-

pha-hydroxy acids, limonene, azone (AZ), lauryl alcohol

(LA), other alcohols, isopropyl myristate (IPM), and so on.

An array of patents is recently focusing on advanced

therapy that uses cells and stem cells that can regenerate

skin’s multi-layered tissue.

Angiogenesis-regulating cells and factors can enhance

wound healing. In a patent, therapeutic platelets are claimed

Table 4. Wound Regeneration Through Gap Junctions Restoration Using a Combination of two Specific Agents [55-61].

Anti-Connexin Agent Structures

First group Anti-connexin oligonucleotides (anti-connexin 43 oligonucleotides), anti-connexin 43 peptides or peptidomimetics, gap junc-

tion closing compounds, hemichannel closing compounds, connexin carboxy-terminal polypeptides

Second group Selected from the above group as modified to subtract the sub-category of anti-connexin agents from which the first anti-

connexin agent was selected.


[65]. Angiogenesis is very important in the early phases of

wound healing, as new blood vessels form in the granulation

tissue. After the healing process is completed, endothelial

cells become quiescent. There is a fine equilibrium between

pro- and anti-angiogenesis processes in which mechanisms

that initiate angiogenesis should trigger, after the healing is

achieved, angiogenesis down-regulation [66]. In first stage

platelets are in increased concentrations triggering wound

healing [67, 68] and proteins-related to this cells can have

pro- and antiangiogenic functions. This patent claims the use

of autologous platelets in order to aid the healing by angio-

genesis regulating factors to the wound site. This patent con-

sists of the delivery in a pharmaceutically carrier of platelets

that have angiogenesis promoting factors [65].

The authors show that the angiogenesis regulating factor

can be selected, from various classes (see Table 6) all factors

activating VEGF pathway.

The platelets can be introduced in a gel that comprises

also the factors that activates angiogenesis regulating factors

(see Table 5). The patent shows that the platelet-rich plasma

(PRP) delivering an efficient healing should comprise plate-

lets in the 1 x 106 - 3 x 10

8 / microliter range of concentra-

tion.

Also in the cell triggering wound healing research do-

main, a recent patent [69] claims the usage of a cell culture

formed by a fibroblast cell feeder layer isolated from skin

tissue. In skin regeneration, one main hurdle is the time pe-

riod required for a full keratinocyte culture. Thus, in this

respect, the patent claims the isolation of fibroblast that have

one of the following cellular membrane markers: ZIC1,

LNX1, EN1, MAFB, HMCN1, LGR4, AGTR1, ITGA1 1,

POSTN, ISLR, DSP, ITGA8, MY01 D, KCNK2, CH25H,

KCNJ15, CLIC2, DPT, TMEPAI, FOXD1, LGR5, SPON1,

HAPLN1, THBS4, CYP26B1, WISP1 and IL-7 [70]. Pub-

lished earlier, a study focusing on the best feeder fibroblasts,

has shown that a sub-population of dermal fibroblasts, iso-

lated from hair bearing skin, can be a better support for

keratinocyte’s growth [71]. Moreover, the prefered anatomi-

cal sites for fibroblast isolation are the scalp, the beard area,

the neck, the arms or the pubic (axilla) region [72]. Interest-

ingly, the patent claims that fibroblast cells isolated from

these anatomical sites gain stemness characteristics as dem-

onstrated in culture by multipotent skin-derived precursors.

The best results for fibroblasts were obtained when fibroblast

were grown on a matrix (e.g. collagen-glucosaminoglycan,

polyhydroxy acids, polyorthoesters, polyanhydrides, pro-

teins, polysaccharides, polyphosphazenes). After initiating

fibroblasts cultures, epidermal cells are grown (e.g. keratino-

cyte, melanocyte, Langerhans or Merkel cell) [73]. These

in vitro cultured tissues, in accordance to the patent, can be

transplanted to patients in order to initiate wound healing

and repair.

In the last years, there were several patents claiming stem

cell induced skin regeneration. Published in 2012 and 2014

[74], these patents claimed processing patient’s adipose tis-

sue regenerative cells intended for wound healing. A new

array of application was opened when adipose tissue was

shown to be a source of stem cells [75, 76]. Moreover, it is

an easy to harvest tissue, can be obtained in large quantities

and the surgery implies low co-morbidities [77]. Cells ex-

tracted from adipose tissue were proven to express angio-

genic growth factors and cytokines (PIGF, VEGF, bFGF,

IGF-II, Eotaxin, G-CSF, GM-CSF, IL-12 p40/p70, IL-12

p70, IL-13, IL-6, IL-9, Leptin, MCP-1, M-CSF, MIG, PF-4,

TIMP-1, TIMP-2, TNF- and Thrombopoetin). Moreover,

these cells would secrete wound healing cytokines (MIP-1

alpha, RANTES, MCP-1, MIG, TARC, MIP-1, KC and

TIMP), would secrete in vitro collagen, and promote in vivo

wound healing. Cells isolated from the adipose tissue have

regenerative potential and mesenchymal stem cells charac-

teristics, hence can promote new vessel formation and

wound healing [78].

The procedure claimed in the patent [79] initiates with

the adipose tissue aspiration, continues with the aspirate be-

ing introduced in a controlled temperature and shaking

Table 5. Inhibitors for Complement Components Claimed in Patent [59].

Complement

System Component

Specific Inhibitor

C3 Compstatin (cyclic peptide, sequence Ile-Cys-Val-Val-Gln-Asp-Trp- Gly-His-His-Arg-Cys-Thr), a compstatin analog, a comp-

statin peptidomimetic, a compstatin derivative, or combinations of these inhibitors

C5a or C5aR Acetyl-Phe-[Orn- Pro-D-cyclohexylalanine-Trp-Arg] (PMX-53), PMX-53 analogs, neutrazumab, TNX-558, eculizumab, pexeli-

zumab or ARC1905, or combinations of these inhibitors

C4 Anti-C4 antibodies

Factor D Diisopropyl fluorophosphates and TNX-234

Factor B Anti-B antibody TA106

Clq Anti-Clq antibodies


chamber in order to start the disintegration procedures and

further isolate regenerative cells. Various disintegration

compounds can be used (neutral proteases, collagenase, tryp-

sin, lipase, hyaluronidase, deoxyribonuclease, Liberase H1,

pepsin, collagenase), or other known physical procedures

like ultrasonic, lasers, microwaves, or other mechanical de-

vices. Buoyant and non-buoyant components are left to settle

and the buoyant layer comprises the regenerative cells that

will be further washed and concentrated. The non-buoyant

layer is to be removed from the disintegration chambers as it

comprises blood, collagen, lipids and other non-regenerative

cells. The cells are washed in sterile condition and the patent

claims that an indicator of red blood removal can be a regis-

tered OD 540nm in the range 0.546 - 0.842, readings that

shows a low contamination with red blood cells. The cell

fraction can contain different types of cells: stem cells, pro-

genitor cells, endothelial precursor cells, adipocytes and

other regenerative cells, this fraction can also contain con-

taminants, such as collagen and other connective tissue pro-

teins. Further filtration is needed to separate only stem cells

or endothelial progenitors cells. The filtration system (e.g.

polysulfone, polyethersulfone or a mixed ester materials)

gradually removes collagen fibers and then larger fibers until

cell suspensions are purified.

Using this method, cell suspensions can be obtained in

the range of 1 105

- 1 107

cells/mL. The automated system

can be re-configurated if the isolated cells need to be sub-

jected to additional manipulation. Thus cells can be further

cultivated and tested for their viability/cell growth, or tran-

siently transfected for further gene therapy applications, or

for generation of cells lines or any other assay development

for cell/tissue engineering applications. When the intended

cells are ready to be used they can be placed into a syringe,

and inoculated subcutaneously, intramuscularly. As stated in

the patent, a portion of the separated batch needs to be cryo-

preserved [79].

From our point of view, we would have expected a stage

were the patent identifyes, in terms of cellular markers, at

least partially, the cell types isolated by this procedure.

Also dealing with mesenchymal stem cells, but this time

derived from skin [80] a patent published in 2013 shows the

methods for isolating, purifying, culturing, storing of three

types of skin-derived cells. The patent focuses on mesen-

chymal stem cells with the differentiating phenotype

CD146+, CD271+ and regeneration-associated cells with the

phenotype SSEA3+ (stage-specific embryonic antigen 3) and

CD105+. From human skin, mesenchymal stem cells

(MSCs) have the phenotype CD146+ and CD271+. CD271

(LNGFR) cells are isolated and these cells can be involved in

the development, survival, and differentiation of regenerative

cells [81]. Primary cells were obtained from a 4mm diameter

adult skin punch biopsy using a previously published method

[82]. From these cells, upon cultivation, cells with specific

markers were sorted using a FACS sorter method. The patent

defines SERA cells as cells expressing SSEA3 and CD105+,

isolated from human dermal skin cells and that can produce

iPSCs. After isolation, SERA cells can be maintained around

8 passages, hence can be cultivated around 6 weeks until

application. These cells can be grown on a scaffold that can

incorporate additional molecules. The scaffold can be biode-

gradable polymeric fibers, collagen fibers, synthetic or natu-

ral extracellular matrix (ECM). Additional molecules can be

active agents such as growth factors, anti-inflammatory

compounds, antibiotics, antivirals, or any intended therapeu-

tical combination.

Like in the previous described patents, in this one cells

can be incorporated in a matrix or in a scaffold in order to

enhance their survival and/or growth. Matrix can be a fibrin

scaffold [83] that can support MSC in a concentration of at

least 1 x 106 cells/mL. This scaffold can have anti-

fibrinolytic agents (tranexamic acid, arginine, lysine) and

anti-coagulation compounds (factor VIII, fibronectin, von

Willebrand factor, vitronectin) [84]. Cells can be mixed with

a hydrogel where cells can adhere and grow, and this combi-

nation can be applied directly [85]. The hydrogel can be de-

signed to fill a cavity that needs to be regenerated. Besides,

the termosensitive hydrogels described above, the patent can

use hydrogels that can be solid in visible or ultraviolet light.

These hydrogels are composed of macromers with a water

Table 6. Angiogenesis Regulating Factors for Wound Healing Mediated by Platelets [65].

Activators For: Compounds

VEGF pathways Agents that activate the neuropilin 1 & 2 pathways, VEGF-A and C, FGF, HGF, angiopoietin-1, insulin-like growth factor-1,

epidermal growth factor, platelet derived growth factor, platelet factor 4, thrombospondin-1, TGF-beta-1, plasminogen activator

inhibitor type-1 (PAI-I), alpha2-antiplasmin and alpha2-macroglobulin VEGFRl (flt-1), VEGFR2 (flk-2), VEGFR3 (flt-4), hepa-

rin sulfate proteoglycan, VEGF121, VEGF145, VEGF165, VEGF168, VEGF189, VEGF -B and -D, PLGF 1, PLGF2, HIV-I

TAT, Sema-E, Sema-III, Sema-IV, bFGF, PDGFR, EGFR, and IGFR

Platelet Thrombin, collagen, serotonin, ADP, acetylcholine and combinations

Angiogenesis PAR-I agonists: TFLLR-NH2; TFLLRNPNDK-NH2; SFLLRNPNDKYEPF-NH2; and SFLLRN-NH2

PAR-4 antagonist: transcinnamoyl- YPGKF-NH2 (tcY-NH2; Ma et al.) and YD-3, a non-peptide PAR4 antagonist nonpeptide

PAR-4 antagonist, YD-3 (ethyl 4-(l- benzyl-lH-indazol-3-yl)benzoate)


soluble region, a biodegradable region, and two polymerizable

regions [86].

The patent [80] describes various utilization for the iso-

lated cells. The direct use in wound repair is far the most

applicable one whether is for initiating and sustaining heal-

ing or for aiding synthetic grafts or autologous skin grafts.

For this later application isolated cells can be added to natu-

ral skin grafts to augment the grafts and can be applied con-

comitantly.

In 2014, a patent was published regarding tissue engi-

neering [87] using autologous dermal fibroblasts combined

with a matrix or scaffold. Fibroblasts are genetically manipu-

lated to secrete a therapeutic protein. Although, the patent is

intended for bone tissue regeneration as the secreted protein

is bone morphogenic protein (BMP-2), the patent claims that

it can be used as well in wound healing. As in previous pre-

sented patent [80] isolated fibroblasts can be incorporated in

a matrix or a scaffold and introduced in the damaged skin

region.

Another extended group of patents focus on the materials

used for skin regeneration, these materials can be new matri-

ces and/or scaffolds for innovative wound dressings. Thus, a

porous keratin material was claimed as a bio-absorbing scaf-

fold that can induce healing. The degree of disulfide cross-

linking between the keratin proteins can vary in relation to

the intended healing capacity [88] for different wound types.

Hence, second degree burns, abrasions, and skin graft have

partial thickness. When these types of wounds are doubled

by co-morbidities like diabetes mellitus or other chronic im-

mune disorders, their healing is hindered. Wounds resulted

from physical injuries, trauma, diabetes leg ulcers, venous

stasis disease, have full thickness with no remaining skin,

and their healing rate is very slow. In wound healing stages,

keratinocytes start the migration from wound edges to the

interior to cover the damaged tissue. This migration is gov-

erned by growth factors (e.g. transforming growth factor- -

TGF- ). Keratin proteins are essential for the re-

epithelization phase. The patent brings as novelty this porous

keratin with controlled degree of disulfide cross-linking

proving a controlled rate of absorption into the wound. This

cross-linking is chemically done by sulfur-sulfur bond estab-

lished between amino acids of adjoining keratin molecules.

The patent claimes that this structure achieves a complete

absorption after 7 days. Keratin is retained in the wound,

aiding the healing process while there is no need to remove

the material from the wound like in other dressing types. The

porous keratin protects the wound medium against oxidative

stress because keratin proteins are one of the richest natural

resources of cystine with anti-oxidative capacity. The mean

degree of cross-linking being in the range of 10- 15%, the

thickness of the porous keratin ranges in the 1 - 3 mm do-

main. An earlier patent described the intact keratin proteins

and keratin protein fractions used in this patent [89].

Biocompatible polymers are subjects for many recent

patents. One published in 2006 described methods for using

fibrin elastomers in dressings for wound repair, implants,

stents, drug encapsulation and drug delivery [90]. The patent

actually describes the process to transform native extracellu-

lar matrix (ECM) molecules, like fibrinogen, in biopolymers.

These biopolymers can incorporate drugs, biological re-

sponse modifiers, antigens, hormones, and other clinically

relevant molecules. Adhesion proteins (collagen, fibronectin,

gelatin, collagen type IV, laminin, entactin, glycosaminogly-

cans, heparan sulfate, RGD peptides, ICAMs, E-cadherins)

can be incorporated to adjoin cells that will trigger wound

healing.

There is a series of patents focusing on blood-derived

plastic materials [91, 92]. This materials can be fibrin,

elastin, natural materials known to be biocompatible [93],

and according to the patents, these materials need to have at

least one cross-linking agent selected from the group pre-

sented in Table 7.

This patent proved that the elasticity of materials ob-

tained herein is 50% - 100% at room temperature. The ob-

tained wound dressings can perfectly seal the wound without

any blood, urine, faces contamination, while maintaining a

moist environment, and, as being sterile, can be designed for

surgical purposes as well [91]. In these materials, biologi-

cally active proteins, sugars, lipids, can be added while their

porosity is controlled to harbor the intended therapeutical

agent [92].

Biomarkers Patents for Wound Healing Evaluations

Patents that are exclusively claiming only healing evalua-

tion biomarkers are few in comparison to those developing

innovative materials. Wound healing process is complex and

comprises three phases: inflammation, proliferation and re-

modeling. These phases have a different time course and

different cells take the leading role in the development of a

particular healing phase. While neutrophils are the predomi-

nant innate immunity cell type during inflammation, fibro-

blasts take the lead in the proliferative phase. The inflamma-

tory reaction is the first response to the wound were granulo-

cytes, mainly neutrophils, monocyte-macrophage lineage are

Table 7. Cross -Linking Agents for Fibroin and Elastin Materials [91].

Type of Cross -Linking Agents

Iridoid derivatives, diimidates, diones, carbodiimides, acrylamides, sugars, proteins, dimethylsuberimidates, aldehydes, factor XIII, dihomo bifunctional

NHS esters, carbonyldiimide, glyoxyls, proanthocyanidin, reuterin


recruited. If this recruitment is normal in the first acute

phases, the uninterrupted presence of neutrophils is associ-

ated with delayed wound healing and chronical status. The

proliferative phase (epithelialization, angiogenesis and ma-

trix formation) starts when neutrophils have been cleared out

by activated macrophages. Hence, in the proliferative phase,

fibroblasts are the main cell-type in a normal healing wound.

Detection of secreted molecules by these types of cells can

be specific biomarkers indicating healing phases and can

indicate wound healing progression. Locally secreted bio-

markers can be used for point-of-care diagnostic determina-

tions [52] (see also Table 1).

In 2014, a report showed that serum C-reactive protein

(CRP) and interleukin-6 (IL-6) evaluated in wound fluid can

depict a local inflammation state of chronic wounds. In this

study the clinical wound improvement was statistically re-

lated to the decrease in wound fluid of these two tested bio-

markers, namely CRP and IL-6 [94].

Patents for Biomarkers. Besides the publications on the

topic of wound healing biomarkers, a patent claimed the anti-

oxidant capacity of a sample to indicate the level of wound

infection. The system comprises the diagnostic device in a

wound dressing that can have also an antimicrobial agent [95].

Symptoms such as local swelling, heat, pain and redness,

is recognized by the physician as infection signs but an ear-

lier detection would be beneficial for patient. Definitive di-

agnosis is routinely achieved by using a swab that extracts

wound fluid and then it is subjected to microbiological tests

that can take at least 48 - 72 hours. This invention claims the

total antioxidant capacity of an infected wound fluid to be

significantly higher compared to a non-infected wound. The

total antioxidant capacity reflects the microbial burden of the

wound. Actually, the wound fluid can inactivate reactive

oxygen species (ROS) such as hydroxyl radicals (OH), sin-

glet oxygen (1O2), hydroperoxyl radicals (

1OOH), superoxide

radical anions (O2 .) and hydrogen peroxide (H2O2). The in-

dicator molecule can be oxidized or reduced by ROS with a

registered modification in absorbance or fluorescence, rou-

tinely a clear colour change. Table 8 describes the redox

indicators that can be used herein.

The antioxidant capacity can be an assayed as the rate of

color change of e.g. cytochrome C due to the superoxide

generation. This method was described more than 50 years

ago [96] and still it is in laboratory use due to its easy to per-

form and robust results. The patent [95] describes the super-

oxide anions generation by the reaction of hypoxanthine with

xanthine oxidase, the generated superoxide anion reacts with

a reference amount of cytochrome C and with the antioxi-

dants present in the wound fluid sample. When evaluating

the activity, the inventors advise that a rise to 130-150% of

the total antioxidant capacity compared to the control can

indicate an infected wound fluid. The wound fluid should be

harvested in the time interval of 1 - 24 hours. Besides the

activity registered, the change in this activity in time should

be as well recorded, evaluating the rate of change. When an

increase in total antioxidant capacity is detected antimicro-

bial dressing is applied.

In 2014, a report showed the healing ability of chronic

ulcers as assessed by evaluating hydroxyproline, total protein

and enzymatic antioxidants (glutathione peroxidase - GPx,

glutathione S-transferase - GST) in the granulation tissue

[97].

Other patent evaluated the stages of wound healing by

quantifing the expression levels of certain genes, biomarkers

for wound healing phases. A chronic wound has as charac-

teristics a reduced level of angiotensin II receptor, IL-1R

receptor antagonist or inositol triphosphate receptor 3, while

the expression of interleukins, growth factors and collagens

is increased. According to the invention, angiotensin II re-

ceptor, IL-1R antagonist, inositol triphosphate receptor 3,

certain interleukins, growth factors and collagens are mark-

ers for wound status. Quantifying by hybridization assay the

RNA extracted from wound tissue and evaluating these ge-

netic markers, the stage of a wound and/or the evolution of

one can be identified [98].

CURRENT & FUTURE DEVELOPMENTS

Wound healing is a wide-spread medical problem and the

history of dressing development has shown that there is no

general all-purpose dressing. Each year, we are witnessing

new materials and procedures whether published and/or pat-

ented. Clinicians have more than 50 different classes of

dressings while 3,000 products aid wound care domain. All

of these products have properties that aim to induce a normal

Table 8. Antioxidant Indicators Used in Patent [95].

Indicator Molecule Target Molecule

Cytochrome C Superoxide

1,10-Phenanthrolene Chelates iron, zinc and other divalent metals

Diphenylamine sulphonic acid Redox indicator

Triphenylmethane dyes Redox indicator

Starch Iodine


wound healing process. Already approved wound dressing

materials are constantly improved, mainly by adding

drugs/growth factors or by down-regulating factors that can

damage the tissue [99]. Although, health care market in

wound management abound, the fact that we still do not

have the best thrapeutical approach for chronic wounds

resides in the lack of cellular and molecular comprehension

of the chronic process.

Advanced approaches in this domain will reside in de-

picting intimate characteristics not only of the wound but as

well for the patient. After establishing these particularities

one can choose the most appropriate, individualized wound

care complex approach. The mechanisms of action for the

wound dressings are very diverse, and while some are effi-

cient in one stage of the wound development, others are

more efficacious in other stage, hence the nurse and the phy-

sician has to develop a specific wound management program

tailored to both wound and the patient carrying it [100, 101].

The future development of patents in wound dressing

field and in the biomarkers domain that enables the assess-

ment of the healing efficiency will move toward the best

physiologically-friendly materials. Innovative scaffolds will

enlarge the incorporated factors and cells that will aid tissue

regeneration. Always keeping in mind that the fate of a

wound resides on both local and systemic approaches, pa-

tient nutrition counseling, education and prevention, should

assist the complex clinical wound management.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-

flict of interest.

ACKNOWLEDGEMENTS

Partially supported by PN-II-PT-PCCA-2013-4-1386

(Project No. 185), PN-II-PT-PCCA-2013-4-1407 (Project

No. 190), PN 09.33-01.01/2009. Author Georgeta Paunica-

Panea has a doctoral grant financed through Project POS-

DRU/159/1.5/S/135760.

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Wound Repair - Updates in Dressing Patents and Regeneration Biomarkers