COMPARISON BETWEEN IONIC SILVER AND PLACENTAL

EXTRACTS AS A LOCAL APPLICATION IN THE MANAGEMENT OF

CHRONIC DIABETIC WOUNDS

S. No Table of Content Page No

1 INTRODUCTION

2 REVIEW OF LITERATURE

3 AIMS & OBJECTIVES

4 MATERIALS & METHODS

5 RESULTS

6 DISCUSSION

7 CONSULATION

8 LIMITATIONS

9 BIBLIOGRAPHY

1

List of Tables

S. No Table Description Page No

1

2

3

4

5

6

7

8

9

10

11

12

2

List of Figure

S. No Figure Description Page No

1

2

3

4

5

6

7

8

9

10

11

12

3

Glossary Abbreviations

4

INTRODUCTION

5

INTRODUCTION

Diabetes mellitus (DM) describes a group of metabolic disorders characterised by high blood

glucose levels. The global prevalence of diabetes and impaired glucose tolerance in adults has

been increasing over recent decades.1 The International Diabetes Federation (IDF) estimated the

global prevalence to be 151 million in 2000, 194 million in 2003, 246 million in 2006, 285

million in 2009, 366 million in 2011, 382 million in 2013, 415 million in 2015 and there are 451

million in 20172, while these figures were expected to increase to 693 million by 2045.2

India is the diabetic capital of the world. As per the recent statistics of IDF nearly 65

million Indians are diabetic of whom more than 8% of the adult population is suffering from

diabetes.2 According to a recent nationwide study by ICMR-INDIAB3 there are large differences

in diabetes prevalence between states in India, with a higher prevalence of diabetes in low SES

groups in the urban areas of the more economically developed states.

People with diabetes have an increased risk of developing a number of serious life-

threatening health problems resulting in higher medical care costs, reduced quality of life and

increased mortality. Persistently high blood glucose levels cause generalized vascular damage

affecting the heart, eyes, kidneys and nerves and resulting in various complications.4 Though,

majority of the patients with diabetes have multiple complications, peripheral neuropathy is the

most common complication in Indian population,5 owing to poor glycemic control.

Diabetic foot is a severe chronic diabetic complication that consists of lesions in the deep

tissues associated with neurological disorders and peripheral vascular disease in the lower limbs.

The incidence of diabetic foot has increased due to the worldwide prevalence of diabetes mellitus

and the prolonged life expectancy of diabetic patients. A latest systematic review found that the

global prevalence of diabetic foot ulcer is about 6.3%6 and that a lower limb is amputated due to

6

diabetes every 30s. The average annual cost of diabetic foot is $8659 per patient7 and the total

medical cost for treating diabetic foot diseases in America ranges from $9 to $13 billion, which

is an additional cost associated with diabetes.8

Diabetic ulcers are known for their chronicity; hence they are a challenge even for an

experienced health care professional. There are many reasons for chronicity of diabetic wounds,

some of which are9, 10

A. Immunopathy: In diabetics, neutrophils are defective, as they have

decreased capacity to migrate to the site of infection and decreased capacity

of phagocytosis and there by intracellular killing of microorganisms. This is

the reason for “polymicrobial infection” commonly found in diabetic

wounds.

B. Neuropathy: Due to sensory neuropathy, there is a decreased perception of

pain which can lead to repeated injuries, late diagnosis of an injury and

injury over a partially healed wound contributes to the chronicity of wound

in diabetics4.

C. Vasculopathy: People with diabetes mellitus have a higher incidence of

atherosclerosis, which contributes to the chronicity by causing local

ischemia and subsequently ulcer formation and their chronicity.

D. Inadequate Level Of Growth Factors: In chronic wounds, level of

growth factors (Platelet Derived Growth Factor, Keratinocyte Growth

Factor) are found to be less. Locally, the levels of proteinases are also

(elastase, matrix metallo proteinases) very high.

7

E. Biofilms: Microorganisms form biofilms readily in diabetics, especially

Pseudomonas aeruginosa. Biofilms give resistance to microorganism from

both antibiotics and phagocytosis2.

F. Other Causes:

1. In diabetics, fibroblasts are found to be defective, which leads to poor

granulation tissue formation.

2. Diabetes leads to psychological stress in patient, so it can release

cortisol which hinders inflammation and forms poor granulation tissue.

3. Uncontrolled blood sugar levels form a microenvironment ideal for

delayed healing and leads the path towards chronicity

The ideal topical agent for use in diabetic wounds should control, prevent infections, help

in formation of good granulation tissue, thereby help in wound healing. Placental extracts helps

in cell migration, collagen matrix formation, and tissue regeneration (fibrogenesis,

neoangiogenesis and epithelization).11 Placental extracts also have bacteriostatic and fungistatic

action. When compared to placental extracts, Ionic silver act mainly by decreasing bio burden at

wound site by bactericidal action. Ionic silver also destabilizes biofilm at wound site, control

infection and help in wound healing.12, 13 The cost of treating a wound with ionic silver is

expensive in a developing country like India, hence placental extracts could offer a low cost but

equally effective modality of treatment, which is feasible in peripheral health settings. However,

there are hardly any studies that compared both of these measures for management of diabetic

8

foot ulcers. So the present study aimed to assess the efficacy between placental extracts and

Ionic silver as topical agents in healing diabetic wounds.

9

AIMS & OBJECTIVES

10

AIMS AND OBJECTIVES:

To compare the efficacy of placental extracts and Ionic silver as topical

agents in healing diabetic wounds.

No use of invasive techniques was proposed.

11

REVIEW OF LITERATURE

12

REVIEW OF LITERATURE

Global burden of Diabetes

The “Diabesity” epidemic (obesity and type 2 diabetes) is likely to be the biggest epidemic in

human history. Diabetes has been seriously underrated as a global public health issue. Currently,

most of the national and global diabetes estimates come from the IDF Atlas. The IDF has

attempted to create awareness of the importance of type 2 diabetes.

The International Diabetes Federation (IDF) estimated the global prevalence to be 151

million in 2000, 194 million in 2003,14 246 million in 2006,15 285 million in 2009, 366 million in

2011,1 382 million in 201316 and 415 million in 2015.17 The World Health Organization (WHO)

also estimated the global prevalence of diabetes in 2000 and 2030–171 million people with

diabetes in 2000 and 366 million by 2030.18

It was estimated that in 2017 there are 451 million (age 18-99 years) people with diabetes

worldwide. These figures were expected to increase to 693 million) by 2045, equaling to 9.9% of

the population, will be living with diabetes. It was estimated that almost half of all people

(49.7%) living with diabetes are undiagnosed. Moreover, there was an estimated 374 million

people with impaired glucose tolerance (IGT) and it was projected that almost 21.3 million live

births to women were affected by some form of hyperglycaemia in pregnancy. In 2017,

approximately 5 million deaths worldwide were attributable to diabetes in the 20-99 years age

range. The global healthcare expenditure on people with diabetes was estimated to be USD 850

billion in 2017.2

In high-income countries, diabetes prevalence peaked (22%) in the 75-79 age group and

in middle-income countries among the 60-74 age groups (19%). In low-income countries, the

prevalence of diabetes peaked (8%) among the 55-64 age group. The prevalence of diabetes

13

among 65-69 year old was 3 times higher in high-income countries compared to low income

countries. Globally, about 79% of people living with diabetes live in low- and middle-income

countries.2

Burden of Diabetes in India

India is considered as diabetic capital of the world. Diabetes is growing alarmingly in our

country. Evidence from available studies suggests that type 2 diabetes in India is a disease of

higher socioeconomic status individuals, and that the diabetes epidemic continues to grow

through conversion from the large pool of individuals with prediabetes. The accuracy of these

assumptions is likely to have changed following the rapid economic development of India over

the past two decades and also due to heterogeneity of the country population in terms of

geography, ethnicity, and sociocultural practices across different states.

The first national study on the prevalence of type 2 diabetes in India was done between

1972 and 1975 by the Indian Council Medical Research (ICMR-New Delhi).19 A National Rural

Diabetes Survey was done between 1989 and 1991 in different parts of the country’s rural

populations which showed diabetic prevalence as 2.8 per cent.20 The prevalence of 6.1 percent in

individuals aged above 40 years was unexpectedly high at that time for rural area with low socio-

economic status and decreased health awareness.21

According to the latest findings from an ICMR-INDIAB study,3 first ever largest

nationally representative, government-funded study of diabetes in India covering all states, the

overall prevalence of diabetes in all 15 states of India was 7·3%, affecting nearly 63 million. The

prevalence of diabetes varied from 4·3% in Bihar to 10·0% in Punjab and was higher in urban

areas (11·2%) than in rural areas (5·2%) and higher in mainland states (8·3%) than in the

14

northeast (5·9%). States with higher per-capita GDP seemed to have a higher prevalence of

diabetes (e.g., Chandigarh, which had the highest GDP of US$ 3433, had the highest prevalence

of 13·6%). In rural areas of all states, diabetes was more prevalent in individuals of higher SES.

However, in urban areas of some of the more affluent states (Chandigarh, Maharashtra, and

Tamil Nadu), diabetes prevalence was higher in people with lower SES. The overall prevalence

of prediabetes in all 15 states was 10·3% (10·0–10·6). The prevalence of prediabetes varied from

6·0% (5·1–6·8) in Mizoram to 14·7% (13·6–15·9) in Tripura, and the prevalence of impaired

fasting glucose was generally higher than the prevalence of impaired glucose tolerance. Age,

male sex, obesity, hypertension, and family history of diabetes were independent risk factors for

diabetes in both urban and rural areas.

Physical complications of diabetes mellitus

Diabetic complications associated with hyperglycaemia impair the metabolism of carbohydrates,

fats, proteins and electrolytes, all of which can disrupt the vascular system.22 Many endothelial

capillary cells are damaged under these conditions, including those in the retina, renal

glomerulus, and both central and peripheral nerves, due to excessive harmful accumulation of

glucose in these cells.23 The critical mechanisms involved in the development of diabetic

complications are mainly induced by chronic hyperglycaemia, impaired lipid catabolism,

exaggerated production of reactive oxygen species (ROS) and a reduced antioxidant protective

system, that all lead to insulin-resistance and increased damage of beta-cells in the pancreas.24

A summary of diabetic complications.

1. Central and peripheral nervous systems

Brain stroke

15

Autonomic neuropathy Peripheral neuropathy (motor and sensory dysfunctions)

2. Eye

Retinopathy Cataracts Blindness

3. Cardiovascular system Cardiomyopathy Myocardial infarction Atherosclerosis Hypertension Endothelial cell dysfunction

4. Oral cavity

• Oral disease (Caries, gingivitis, periodontal abnormalities, infections)

5. Renal system Nephropathy Proteinuria Glucosuria Kidney failure

6. Gastrointestinal system Delayed gastric emptying Diarrhoea Constipation Dyspepsia Exocrine gland insufficiency

7. Genital system

Impotence Sexual dysfunction Urogenital dysfunction

8. Skin and soft tissues

16

Wound healing impairment Skin infection

9. Bone

Osteopenia, fractures

10. Foot

Foot ulceration Foot amputation

Diabetic foot disease: global and Indian burden

Diabetic foot is a severe chronic diabetic complication that consists of lesions in the deep tissues

associated with neurological disorders and peripheral vascular disease in the lower limbs. The

incidence of diabetic foot has increased due to the worldwide prevalence of diabetes mellitus and

the prolonged life expectancy of diabetic patients. A previous study showed that a lower limb is

amputated due to diabetes every 30s,25 and the average annual cost of diabetic foot is $8659 per

patient.26 Of all amputations in diabetic patients, 85% are preceded by a foot ulceration which

subsequently deteriorates to a severe gangrene or infection.25

Global burden

A recent systematic review and meta-analysis revealed that the pooled worldwide prevalence of

diabetic foot ulcers (DFU) was 6.3%, with Belgium reporting the highest prevalence of 16.6%

while Australia and New Zealand having the lowest prevalence of 3%.6 The prevalence in Africa

was 7.2%, which was higher than Asia 5.5% and Europe 5.1%.

The prevalence of diabetic foot ulceration from hospital-based (7.1%) and public health

center studies (5.6%) was higher than from population-based (4.6%) and community-based

(2.9%) studies. Gender wise, diabetic foot ulceration was more prevalent in male diabetic

patients (4.5%) than female patients (3.5%).6 Diabetic foot ulceration was also more prevalent in

17

patients with type 2 diabetes mellitus (6.4%) than in patients with type 1 diabetes mellitus

(5.5%).

When compared to the diabetic patients without foot ulcers, the patients with foot ulcers were of

older age, longer diabetic duration, lower body mass index, higher percentage of smokers, with

hypertension and diabetic retinopathy.

Burden of diabetic foot ulcers in India

India has one of the highest prevalence of foot ulcers among diabetics (15%), while a previous

meta-analysis found the prevalence to be 11.6%.6 Evidence from published literature showed

100,000 leg amputations/ year due to diabetes-related problems and an expense of approximately

$1,960 for complete treatment of DFUs. Out of 62 million diabetics in India, 25% develop

DFUs, of which 50% become infected, requiring hospitalization while 20% need amputation.27

DFUs contribute to approximately 80% of all non-traumatic amputations in India, annually.

Patients with a history of DFU have 40% higher 10-year death-rate, than those without. Average

time required for healing of DFUs is 28 weeks (range 12-62 weeks). Also India is the most

expensive country for DFU care as 5.7 years of an average patient’s income is required to pay for

the complete DFU therapy.27

18

Pathophysiology of DFD

Why diabetic wounds delay in healing?

Diabetic wounds pose a challenge to even to most experienced health professionals. There are

many factors responsible for delayed healing and chronicity of wounds:

Figure 1: factors contributing for delayed wound healing in diabetics

Immunopathy in Diabetes

Infections are increasingly prevalent in diabetic patients due to immune system deficiency.

Diabetics have impaired polymorphonuclear function with decreased capacity to migrate to the

site of infection, decreased capacity to phagocytosis.28 A significantly lower chemotaxis has been

found in polymorphonucleocytes of diabetic patients (type 1 and type 2) than in those of

controls.29-31 The mean HbA1c concentration was lower (better regulation) in patients without

impaired phagocytosis29 than in those with impaired phagocytosis.32 Polymorphonucleocytes of

diabetic patients have shown lower phagocytic capacity compared to polymorphonucleocytes of

controls.

19

DELAYED WOUND HEALING

WOUND INFECTIONS

ISCHEMIA

FAULTY WOUND HEALIN

G

Diabetic neuropathy

Chronic HG may lead to either sensory or motor neuropathic problems or autonomic nervous

system dysfunction. However, patients with long-term diabetes may have one or more types of

neuropathies.

Peripheral neuropathy:

Diabetic peripheral neuropathy is one of the major com- plications affecting patients with DM.

This can lead to either sensory or sensorimotor neuropathies that increase the risk of foot

ulceration and amputation in some cases of uncontrolled diabetic patients.33

In diabetics there will be decreased sensitivity to pain, leading to small wounds due to

trauma. These wounds later gets infected and become chronic. Initially when patient get injured,

they do not notice wound as it is not painful. This makes wound more prone to get infected.

Localized pressure also plays major role in diabetics, for example bed sore, infected corn in the

foot, if not treated properly can progress into chronic ulcer.

Vasculopathy in Diabetics

Diabetics will have micro and macroangiopathy. Decreased oxygenation and ischemia activates

inflammation at the site of wound that attracts neutrophils. Neutrophils release inflammatory

cytokines, proteolytic enzymes, reactive oxygen species (ROS) which damage cells at wound

site, prevent proliferation and wound healing. Neutrophils stay for longer time in diabetic

wounds compared to other acute wounds, leading to fact that chronic diabetic wounds have

higher levels of inflammatory cytokines and reactive oxygen species (ROS).34

20

Vascular complications in DM is classified into two categories, namely macrovascular,

which includes coronary and peripheral arterial disease, and microvascular, which is associated

with other DM-induced long-term complications such as neuropathy, retinopathy, nephropathy

and in part, diabetic foot and cardiovascular diseases.35

The endothelial cell impairments involved in macrovascular complications have many inducing

elements, including elevated blood glucose levels, MGO, lipids, and inflammatory factors.36 DM

is also associated with excessive pro- duction of ROS, which in turn can induce vasoconstriction

with accelerated lipid peroxidation and inflammatory reactions leading to atherosclerosis.35

Inadequate levels of growth factors:

In chronic wounds, compared to acute wounds the levels of proteolytic enzymes (elastase, matrix

metallo-proteinases) are high, while the concentration of growth factors (platelet derived growth

factor, keratinocyte growth factor etc.) are low. Inadequate growth factors play a major role in

defective wound healing in diabetics.37

21

Figure 2:

Bacteria and biofilms

In diabetics chronic wounds affects patients quality of life more than vision loss or renal failure.

Recently alteration in skin microbiota is emerging to have an impact on delayed healing of

diabetic wounds. Diabetic wounds will have high microbial burden. 85% of amputations in

patients with diabetes are preceded by infected wounds. Diabetic ulcers are polymicrobial and

multi drug resistant with ability to form biofilms. Biofilm forming bacteria are 1000 times more

22

resistant to antibiotics.38 So biofilm formation is a very important virulence factor and main

reason for treatment failure. Most common organism that infect chronic diabetic foot ulcer is

Staphylococcus aureus, followed by Escherichia coli, Pseudomonas aeruginosa, Citrobacter

species, Klebsiella oxytoca, Proteus respectively.39 Among the above mentioned bacteria

Staphylococcus aureus is the most common biofilm former followed by Pseudomonas

aeruginosa and Citrobacter.

Evidence suggests that nearly 80% of bacterial species isolated from diabetic foot ulcers

were multi drug resistant.38 Most commonly used antibiotic is amoxicillin + clavulanic acid

followed by clindamycin. 50% of gram negative bacterial organisms show resistance to

amoxicillin + clavulanic acid. Imepenum, piperacillin + tazobactum, cefaperazone + sulbactum

reported as most effective drugs against diabetic foot ulcer infections.

Biofilm structure has been analyzed microscopically and biochemically. Biofilm is made

up of multilayered matrix containing water, bacterial cells, proteins, DNA and polysaccharides.

Figure 3: Composition of Biofilm.

Close cell to cell contact in biofilms always allow easy transfer of plasmid containing multi

drug resistance (MDR) genes amongst one another. Organisms which form biofilms are also

characterized by tolerance which is temporary and non-heritable character. Mechanism for

tolerance:

23

a) Antibiotics that prevent cell division are ineffective against organisms producing

biofilms.

b) Drug penetration is hindered by polysaccharide matrix.

c) Drug efficacy is altered by pH of microenvironment of biofilm.

d) Biofilms hide microorganisms from host defense

Faulty fibroblasts

Fibroblasts in diabetics have a reduced capacity to produce extra cellular matrix proteins and

keratinocytes that epithelize the wound.

Epithelial progenitor cells (EPC’s) which are derived from bone marrow travel to the site

of injury and help in formation of blood vessels and wound healing. It has been revealed that

EPC’s are essential for wound healing are decreased both in circulation and at wound site and

that impaired nitric oxide synthase (NOS) activation and decreased stromal cell derived factor - 1

alpha (SDF-1 alpha) at wound site could be the main reason for impaired wound healing.40, 41

In diabetics every scratch on skin is a matter of concern, as they have impaired wound

healing. Every small wound has potential to become infected chronic wound leading to sepsis,

amputation and even death.

Other causes:

Many factors affect wound healing in diabetics such as age, comorbidities, Wound etiology, size

of wound, location of wound, heavy bio burden, nutritional status etc. stress also plays a major

role in wound healing. Diabetics with non-healing wounds are at tremendous stress which will

have negative effect on wound healing. Stress increase cortisol levels that lowers immunity and

inflammation, thereby increase chance of infections.42 Co-morbid conditions always contribute

24

for wound to become chronic in Diabetics e.g. diabetics with chronic disease are more prone to

methicillin resistant staphylococcus aureus (MRSA).

Figure 4: multiple factors contributing to diabetic foot ulcers

Impact of delayed wound healing

Chase et al.43 introduced the concept of “forever healing and permanent wounding” in diabetics.

Patients with chronic diabetic wounds will have poor quality of life. It involves loss of time

because of multiple hospital visits over months, time off work or loss of employment with

significant financial burden on patient and his family. Also affects the social wellbeing of the

patient. Diabetic wounds resulting in amputation increase threefold risk of death in next 18

months to 2 years.44 Diabetic wounds are most common cause of disabling chronic and expensive

complication of diabetes.

25

Figure 5: Increased chance of mortality after amputation in diabetics

Management of chronic diabetic wounds

It is estimated that 15% of diabetes patients will develop Diabetic foot ulcer once in their life

time, and approximately 14% of diabetic ulcers lead to amputation unless a prompt, rational,

multidisciplinary approach to therapy is taken. Factors that affect development and healing of

diabetic patient’s foot ulcer include the degree of metabolic control, the presence of ischemia or

infection, and continuing trauma to feet from excessive plantar pressure or poorly fitting shoes.

Appropriate wound care for diabetic patients addresses these issues and provides optimal local

ulcer therapy with debridement of necrotic tissue, provision of a moist wound healing

environment and applying topical agents locally that help in healing. During the prolonged

healing process of a chronic wound, rapid and accurate evaluation of the healing progress is

26

critical so that unsuccessful treatments can be discontinued and alternate treatments be initiated

as soon as possible.

The “TIME” framework in diabetic wound management encompasses tissue management,

inflammation and infection control, moisture balance, and epithelial (edge) advancement.24

Tissue management aims to remove the necrotic tissue burden via various methods of

debridement. Infection and inflammation control by reduction of bacterial biofilms, antibiotic

use, local topical agents, Achieving a moist wound healing environment without excessive

wound moisture or dryness will result in moisture balance. Epithelial advancement is promoted

via removing the physical and biochemical barriers for migration of epithelium from wound

edges. These systematic and holistic approaches will potentiate the healing abilities of the

chronic diabetic ulcers, including those that are recalcitrant.

Figure 6: “TIME” acronym for management of chronic diabetic wounds.

27

Adequate debridement, application of topical wound healing agents and dressings is

needed for chronic diabetic ulcers. Many techniques are available for wound dressing and all

essentially reduce infection and improve granulation tissue growth. In this study the wound

healing potential of topical placental extract was evaluated and compared with topical ionic

silver.

Importance of placental extracts in chronic diabetic wounds

It is known from traditional folk knowledge that the placenta, supporting the baby's growth and

development in the mother’s womb, contains a wide range of biologically active components.

Research over decades has been uncovering more and more of these compounds. Indeed, it is

claimed that the placenta is capable of producing just about any substance found in any organ of

the body.

Placenta growth factor is a dimeric glycoprotein, structurally and functionally related to

the vascular endothelial growth factor, a potent angiogenic/permeability factor known to play a

role in the neoangiogenesis during wound repair.

This biochemical treasure house supplies the growing foetus with substances that the

foetus itself cannot synthesize. Though a rich source of bioactive components unless recovered,

placenta becomes a biomedical waste immediately after childbirth. Use of human placenta as a

therapeutic agent in no way hampers ecological balance rather promotes resource recovery from

a designated biomedical waste. Research on human placental extract gained a momentum with

the description of the preparation of its extract by Russian ophthalmologist Prof. V.P. Filatov, he

used placental extracts in grafting human corneas in the year 1912.

28

Placenta serves as a natural storehouse of many biologically active components with

significant healing attributes. It actually involves in almost every stage of healing. Only aqueous

form is biologically active. Modern indigenous aqueous placental extract is prepared employing

Filatov’s procedure. Fresh placenta retrieved after baby’s birth is tested for HIV, HBsAg and

HCV. Single hot and cold aqueous extractions were done after incubating placenta at 90oC and

6oC respectively. This was followed by sterilization of extract under saturated steam (pressure

15-lbs/sq. inch at 120oC for 40 min). After filtration and addition of 1.5% benzyl alcohol as

preservative, ampoules were filled and sterilized once again under the said condition for 20

minutes.45 Each millilitre of drug was derived from 0.1 g of fresh placenta. A single batch was

prepared from pool of several placentae.

29

Figure7: Cellular mechanism of wound repair in placental extract therapy.

Mechanism of action of placental extract in healing wounds

Aqueous extract of placenta contains nucleotides like Polydeoxyribonucleotides (PDRNs),

known for their regenerative effect.46 Placental extract is rich in enzymes like alkaline and acid

phosphatases, glutamic oxaloacetic acid transaminase; RNA, DNA, and ATP; Vitamins like B1,

30

B2, B6, pantothenic acid, biotin, Folic acid, B12, choline, and inositol; Amino acids like alanine,

aspartic acid, cysteine, histidine, leucine, phenylalanine, proline, serine, threonine, tryptophan,

valine and tyrosine; steroids; elements like Na, K, Ca, Mg, Cu, Fe, P. All these components may

exert multiple biological activities. Placental extract also has Corticotropin Releasing Factor

(CRF) which is proven to be promoter of Human Epidermal Keratinocyte Proliferation.47

Placental extract increases collagen synthesis, increases tissue protein, accelerates

neoangiogenesis, and epithelialization. Has immunotropic effect on EGF (Epidermal Growth

Factor) and Fibroblast growth factor. It reduces surrounding tissue inflammation and edema.

Studies have shown that use of topical placental extract increases the rate of wound healing and

patients have an early recovery.48

Chakraborty et al. studied the role of placental extracts on the growth of different bacteria 26.

They found that placental extracts prevents the growth of bacteria such as E.coli from urine and

blood culture. They found placenta to also have an inhibitory role in the growth of bacteria such

as Staphylococcus aureus, fungi such as Saccharomyces cerevisae, Kluyvero-myces fragilis, and

Candida albicans. Sudhir et al studied the effect of topical placental extract dressing over various

diabetic ulcers and had similar results.11 No side effect has been noted with placental therapy.

Importance of Ionic silver in chronic Diabetic wounds

Silver is a precious metal, its medicinal properties were known from ages (over past 200 years)

as silver coins and vessels were used for drinking water purification. Most of the diabetic

31

wounds are infected. Topical ointments containing ionic silver are used to prevent and manage

infection in wide range of wounds. The topical antimicrobial agent silver has been used for

hundreds of years in wound care. For example, silver has been used to prevent or manage

infection in its solid elemental form (e.g. silver wire placed in wounds), as solutions of silver

salts used to cleanse wounds (e.g. silver nitrate solution), and more recently as creams or

ointments containing a silver–antibiotic compound (silver sulfadiazine (SSD) cream).

Silver is found in a number of forms49

■ Elemental silver: e.g. Nano crystalline silver.

■ Inorganic compound: e.g. silver oxide, silver phosphate, silver chloride, silver-calcium-

sodium phosphate, silver zirconium compound, Silver sulfadiazine.

■ Organic complex: e.g. silver-zinc allantoinate, silver alginate, silver carboxymethylcellulose.

Mechanism of action of silver at wound site

In metallic (elemental) form, silver is unreactive and cannot kill bacteria. To become

bactericidal, silver atoms (denoted as Ag ) must lose an electron and become positively charged

silver ions (Ag+). Elemental silver ionizes in air, but ionizes more readily when exposed to an

aqueous environment such as wound exudate. Silver ions are highly reactive and affect multiple

sites within bacterial cells, ultimately causing bacterial cell death. They bind to bacterial cell

membranes, causing disruption of the bacterial cell wall and cell leakage. Silver ions transported

into the cell disrupt cell function by binding to proteins and interfering with energy production,

enzyme function and cell replication.50 Silver ions are active against a broad range of bacteria,

fungi and viruses, including many antibiotic-resistant bacteria, such as meticillin-resistant

Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE).51 Ionic silver

32

reduce bacterial adhesion and destabilize the biofilm matrix, as well as kill bacteria within the

matrix and increase susceptibility of bacteria to antibiotics.13 Ionic silver have be found to have

anti-inflammatory effect, increase neovascularization at wound site.52

Antibiotics act only at single site on target bacterial cell, which is the main reason for

increasing antibiotic resistance. Compared to antibiotics, ionic silver acts at multiple sites on a

target cell.50 Therefore, chance of developing resistance to ionic silver is less. So it can play a

potential role in controlling infection and limiting antibiotic use, thereby decreasing further

chance of developing antibiotic resistance.

The aim of treatment with silver dressings is to reduce wound bioburden, treat local

infection and prevent systemic spread. Their main purpose is not to promote wound healing

directly. Clinical guidelines recommend that silver dressings are used for wounds where

infection is already established or an excessive wound bioburden is delaying healing.53, 54 Silver

dressings should not be used on wounds where bioburden is not a problem, i.e. they should be

reserved for use in wounds with or at risk of high bioburden or local infection.55

33

Figure 8: Mechanism of action of ionic silver on bacterial cell- attachment to the bacterial

cell wall, its diffusion into bacterial cell and coagulating bacterial proteins/enzymes.

Silver dressings occasionally cause local skin discoloration or staining which is harmless

and usually reversible. This discoloration is not true systemic argyria, which is rare and usually

related to oral ingestion of silver solutions as an alternative health practice. So ionic silver

preparations are non-toxic and few studies even found that ionic silver promotes wound healing

in chronic wounds.56 Studies show ionic silver have antimicrobial activity against a wide range of

microorganisms, including resistant forms such as MRSA and VRE, and fungi and anaerobes.50

34

Most relevant studies

1. Carotti, D and Allegra, E in 1981 described a procedure for characterization of the

proteinic, peptidic, and amino-acidic components of human placental extracts. Four

different preparations were analysed. The first was an extract prepared in our laboratory

from fresh on-term placenta. The other three were commercial products obtained by

different procedures including autolysis and sterilization. The patterns given by each type

of preparation are reproducible and characteristic.48

2. O'keefe et al. in 1985 tested extracts of term human placenta for enhancement of

proliferative growth of primary cultures of human keratinocytes. Saline extracts or

supernatants from homogenates were dialyzed extensively, lyophilized, and tested in

subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only

defined supplements (insulin, hydrocortisone, transferrin, ethanolamine,

phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities

(1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract

at 25-500 micrograms/ml. Extracts of cord serum or maternal serum were inactive,

suggesting that the activity is derived from placental tissue. The presence of activity in

the placenta with distinctive properties suggests that this is a previously undescribed

material with growth-promoting properties for epithelium.47

3. Failla et al. in 2000 evaluated the expression of placental growth factor (PlGF) in human

keratinocytes and investigated its possible role in wound healing. Northern blot analysis

on cultured keratinocytes revealed a 1.7 kb mRNA transcript and reverse transcriptase-

polymerase chain reaction allowed the detection of two PlGF isoforms generated by

35

alternative RNA splicing. The analysis of human full-thickness healing wounds revealed

appreciable levels of PlGF mRNA and protein in the migrating keratinocytes starting

from day 3 after injury, and increasing at day 5. The data demonstrated that keratinocytes

are a source of PlGF during wound healing in vivo and indicate a role for this factor in

the neoangiogenesis process associated with cutaneous wound repair.57

4. Lansdown AB in 2002 in their first in a two-part series described the antimicrobial

properties and mechanism of action of silver. Silver products have two key advantages:

they are broad-spectrum antibiotics and are not yet associated with drug resistance. The

reviewed literature revealed that the microbicidal action of silver products has been

directly related to the amount and rate of silver released and its ability to inactivate target

bacterial and fungal cells. Chemically, metallic silver is relatively inert but its interaction

with moisture on the skin surface and with wound fluids leads to the release of silver ion

and its biocidal properties. Silver ion is a highly reactive moiety and avidly binds to

tissue proteins, causing structural changes in bacterial cell walls and intracellular and

nuclear membranes.50

5. Chauhan et al. in 2003 compared two topical agents, placental extract and phenytoin

powder on 150 patients, with non-healing ulcers. They were randomly assigned to these

treatments or to saline dressings (control). It was observed that patients receiving active

topical treatments responded better than those in the control group. The importance of

this finding should be viewed with the perspective that these topical treatments are

inexpensive and easily available in India. The study also piloted measurements of

angiogenic responses in 1 group, and the findings encourage further exploration with the

technique and topical agent.58

36

6.Coutts P and Sibbald RG in 2005 evaluated the clinical improvement in chronic wounds

through the effect on wound size, maceration, resolution of surface slough and

conversion to healthy granulation during a 4-week application of the silver-containing

Hydrofiber dressing. This was a single centre, open-label case series study which

included a total of 30 evaluable participants: four with diabetic neuropathic foot ulcers,

13 venous stasis ulcers, four pressure ulcers and nine miscellaneous wounds that did not

fit any of the previous categories. All participants had adequate vascular supply,

indicating the potential to heal. The underlying cause of the ulceration was identified and

corrected, or the symptoms and signs were treated. This was followed by the application

of silver-containing Hydrofiber dressings for a period of 4 weeks. The majority of

wounds treated decreased in size (70%) with decreased exudate, decreased purulence and

resolution of surface slough (75%). There was an increased quality and quantity of

healthy granulation tissue. Unlike some silver dressings, the Hydrofiber and silver

combination dressing was unlikely to cause burning and stinging on application.59

7.Jorgensen et al. in 2005 compared the effect of a sustained silver-release foam dressing

(Contreet Foam) with a foam dressing (Allevyn Hydrocellular) without added silver in

critically colonised venous leg ulcers with delayed healing through a multicentre, open,

randomised, controlled study lasting for 4 weeks. Ulcer area and healing were assessed

weekly. Odour, maceration, absorption capacity and leakage were evaluated at dressing

changes. All adverse events were recorded. One hundred and twenty-nine patients were

included (Contreet Foam: 65, Allevyn Hydrocellular: 64) where in the two groups were

comparable in all respects. After 4 weeks, there was a significantly greater reduction in

ulcer area in the Contreet Foam group (45%) than in the Allevyn Hydrocellular group

37

(25%). There was evidence of the superior performance of the silver-releasing dressing,

Contreet Foam, compared with a traditional moist foam wound healing dressing in the

treatment of critically colonised, chronic venous leg ulcers.60

8. Chakraborty et al. in 2005 described about isolation of fibronectin type III like peptide

from human placental extract used as wound healer. A peptide of around 7.4 kDa has

been purified from the aqueous extract of human placenta used as wound healer. Derived

partial amino acid sequence from mass spectrometric analysis showed its homology with

human fibronectin type III. Under nondenaturing condition, it formed aggregate, the

elution pattern of which from reverse-phase HPLC was identical with that of fibronectin

type III. Immuno-blot of the peptide with reference fibronectin type III-C showed strong

cross reactivity. They opined that since fibronectin type III plays important roles in

wound healing, similar peptide in the extract is likely to take part in curing process.11

9. Cianfarani et al. in 2006 assesed the altered expression and therapeutic potential of

placental growth factor in diabetic wound healing by using streptozotocin-induced

diabetic mice. They observed that the PlGF induction is strongly reduced in diabetic

wounds. Diabetic transgenic mice overexpressing PlGF in the skin displayed accelerated

wound closure compared with diabetic wild-type littermates. Moreover, diabetic wound

treatment with an adenovirus vector ex- pressing the human PlGF gene (AdCMV.PlGF)

significantly accelerated the healing process compared with wounds treated with a

control vector. They concluded that reduced PlGF expression contributes to impaired

wound healing in diabetes and that PlGF gene transfer to diabetic wounds exerts

therapeutic activity by promoting different aspects of the repair process.61

38

10. Bergin SM and Wraight P in 2006 did a systematic review to evaluate the effects of

silver-containing dressings and topical agents on infection rates and healing of diabetes

related foot ulcers. Searches were made of the Cochrane Wounds Group Specialised

Register, the Cochrane Central Register of Controlled Trials, MEDLINE (1966 to

October week 2 2004), EMBASE and CINAHL. Randomised controlled trials and non-

randomised controlled clinical trials were considered for inclusion. Studies were included

if they involved participants with Type 1 or Type 2 diabetes and related foot ulcers, met

the requirements for randomisation, allocation and concealment where appropriate, and

compared the intervention with a placebo or a sham dressing, an alternative non silver

based dressing or no dressing, and reported outcomes that represent healing rate or

infection. Two authors independently evaluated the papers identified by the search

strategy against the inclusion criteria but identified no trials eligible for inclusion in the

review. It was concluded that despite the widespread use of dressings and topical agents

containing silver for the treatment of diabetic foot ulcers, no randomised trials or

controlled clinical trials exist that evaluate their clinical effectiveness. Trials are needed

to determine clinical and cost-effectiveness and long term outcomes including adverse

events.62

11. Leaper DJ in 2006 reviewed on the role of silver dressings in wound healing. They noted

that although silver has been recognised for centuries to inhibit infection its use in wound

care is relatively recent. Evidence of the efficacy of the growing number of silver

dressings in clinical trials, judged by the criteria of the Cochrane Collaboration, is

lacking, but there are good indications for the use of silver dressings, to remove or reduce

an increasing bioburden in burns and open wounds healing by secondary intention, or to

39

act as a barrier against cross contamination of resistant organisms such as MRSA. More

laboratory, and clinical data in particular, are needed to prove the value of the many

silver dressings which are now available. Some confusion persists over the measurement

of toxicity and antibacterial activity but all dressings provide an antibacterial action,

involving several methods of delivery. Nanocrystalline technology appears to give the

highest, sustained release of silver to a wound without clear risk of toxicity.56

12. Cutting et al. in 2007 did a review on the safety and clinical efficacy of silver dressings.

They noted that the use of silver extensively as a medicament raises concerns, which

centre on issues such as resistance and toxicity, clinical efficacy and cost-effectiveness.

The silver-containing dressing segment of the medical device market is of huge

commercial importance, and, consequently, marketing and promotional issues

occasionally obscure the evidence that clinicians need to have in order that they may

provide appropriate treatment for their patients. The impact of silver application on the

wound bioburden needs to be examined carefully to heighten our awareness of any

deleterious effects on the healing process, without inducing any unfounded anxieties.53

13. Jude et al. 2007 did a prospective, multicentre study compared clinical efficacy and

safety of AQUACEL Hydrofiber dressings containing ionic silver (AQAg) with those of

Algosteril calcium alginate (CA) dressings in managing out-patients with Type 1 or 2

diabetes mellitus and non-ischemic Wagner Grade 1 or 2 DFUs. Patients stratified by

antibiotic use on enrolment were randomly assigned to similar protocols including off-

loading, AQAg (n = 67) or CA (n = 67) primary dressings and secondary foam dressings

for 8 weeks or until healing. Clinical efficacy measures were healing outcomes and

primarily healing speed. Adverse events were recorded. When added to standard care

40

with appropriate off-loading, AQAg silver dressings were associated with favourable

clinical outcomes compared with CA dressings, specifically in ulcer depth reduction and

in infected ulcers requiring antibiotic treatment. This study reports the first significant

clinical effects of a primary wound dressing containing silver on DFU healing.63

14. Vermeulen et al. in 2007 carried out a systematic review and meta-analysis to assess the

effects on wound healing of topical silver and silver dressings in the treatment of

contaminated and infected acute or chronic wounds. We sought relevant trials from the

Cochrane Central Register of Controlled Trials (CENTRAL), the Cochrane Wounds

Group Specialised Register in March 2006 and in MEDLINE, EMBASE, CINAHL, and

digital dissertations databases up to September 2006. In addition, we contacted

companies, manufacturers and distributors for information to identify relevant trials.

Randomised controlled trials (RCTs) assessing the effectiveness of topical silver in the

treatment of contaminated and infected acute or chronic wounds. Eligibility of trials,

assessment of trial quality and data extraction were undertaken by two authors

independently. Disagreements were referred to a third author. Only three trials with a

short follow-up duration were found. There is insufficient evidence to recommend the use

of silver-containing dressings or topical agents for treatment of infected or contaminated

chronic wounds.64

15. Woo et al. in 2008 wanted to ascertain the best practices whether to prefer SILVER or

other antimicrobial. They opined that in view of the ubiquitous presence of microbes, the

clinician must discern whether bacterial balance (contamination or colonization) or

bacterial damage has occurred. Silver was a common topical agent used to combat

bacterial burden in chronic wounds. Given the wide array of silver-related wound care

41

products, it was difficult to determine which product should be used. By reviewing

relevant scientific evidence, we propose an acronym SILVER to address the key

contentious issues. These issues were summarized as SILVER: Signs of bacterial

damage, the need for Ionic silver, Log reduction of bacteria, Vehicle (importance of

moisture balance), Effect on normal cells, and Bacterial Resistance.49

16.

MATERIALS & METHODS

42

Study site: This study was conducted in the Department of general surgery, St Isabel’s hospital,

Chennai.

Study population: All the eligible patients attending Department of General Surgery, St Isabel’s

hospital, over a period of 2 years (April 2016 till March 2018), satisfying the inclusion/exclusion

criteria were considered as study population.

Study design: The current study was a prospective, randomized, comparative study.

Sample size:

μ1 39.00μ0 29.00u 1.28

v 1.96

σ1 12.00σ0 11.00

27.82 in each group

Sampling method: All the eligible subjects were recruited into the study consecutively by

convenient sampling till the sample size is reached.

Study duration: The data collection for the study was done between April 2016 to March 2018

for a period of 2 year.

Inclusion Criteria:

• Patients with more than 15yrs of age.

• Patients with diabetic wound of more than 3wks of duration.

• Serum albumin more than 3g/dl.

43

(u + v)2 (σ12 + σ02)(μ1 - μ0)2

Exclusion criteria:

• Patients with non-diabetic ulcers.

• Patients who have ulcer in diabetics due to a known other etiology (example: venous

ulcers in patients with varicose veins).

Ethical considerations: Study was approved by institutional human ethics committee. Informed

written consent was obtained from all the study participants and only those participants willing to

sign the informed consent were included in the study. The risks and benefits involved in the

study and voluntary nature of participation were explained to the participants before obtaining

consent. Confidentiality of the study participants was maintained.

Data collection tools: All the relevant parameters were documented in a structured study proforma.

Methodology:

It was a prospective, randomized, comparative study in which patients who

satisfy inclusion and exclusion criteria were recruited after explaining about the study and

getting an informed consent. After recruiting into study patients name, age, sex, duration of

diabetes, Hba1c, serum albumin, other comorbidities were noted. Data about history of smoking

and alcohol was noted.

Patients with diabetic wound were placed on ionic silver (containing 32ppm of silver

ions) or on placental extracts (strength 0.25%/0.1g) randomly by random number tables and at 1st

visit data about wound (site, size, duration of wound, presence of slough and dead tissue,

presence of pus) were collected and at 1st visit itself wound swab was sent for culture and

sensitivity. Wounds with dead and necrotic tissue were thoroughly debrided and topical

ointment applied according to group in which patient belongs to.

Clinical, laboratory parameters and operative details were analysed. Patients were

followed up and wounds were reassessed end of 1st week, end of 4th, end of 6th week, end of 9th

week, and at end of 12th week. On other days they were instructed to get dressing done at home

44

with prescribed topical application. At follow up wound was reassessed and size of wound,

percentage reduction in size of ulcer, percentage of ulcer remaining, presence of slough and

necrotic tissue, presence of pus, time in days taken for complete granulation tissue were noted.

END POINT:

At every follow-up visit the size of the wound was measured.

• Before or at end of 12 weeks wounds that are completely healed are taken as healed cases

TREATMENT FAILURE:

• wounds increased in size by the end of 4weeks from starting treatment or

• Less than 50% decreased in size/no decrease in size at end of 6 weeks of starting

treatment or

• Initially after 6wks there was more than 50% decrease in size of wound and from there

wound increase in size and at end of 12weeks wound not completely healed.

In the above case scenarios, the treatment was considered as failure. We abandoned the study

procedure and there on the patient was given other available treatments. For treatment failure

cases at the last review tissue repeat culture from wound was done to know which

microorganism was responsible for nonhealing and treatment failure in that wound.

STATISTICAL METHODS:

Topical ointment (placental extract, ionic silver) was considered as primary explanatory variable.

variable.

Area of the ulcer, percentage reduction in ulcer area and final healing status were considered as

primary outcome variables.

 Age, gender, duration of diabetes mellitus (in years), CBG on 1st visit, Hba1c (%),plasma

albumin (gm%), etc were explanatory variable.

Descriptive analysis was carried out by mean and standard deviation for quantitative variables,

frequency and proportion for categorical variables.

The association between topical ointment group (placental extract, ionic silver) and quantitative

outcome was assessed by comparing the mean values. The mean differences along with their

45

95% CI were presented. Independent sample t-test was used to assess statistical significance. .

Data was also represented using appropriate diagrams like error bar diagram.

The association between topical ointment group (placental extract, ionic silver) and categorical

outcomes was assessed by cross tabulation and comparison of percentages. . Data was also

represented using appropriate diagrams like cluster bar diagram.

Normality test for quantitative variables:

A shapiro- wilk’s test (p>0.05) 65, 66 and a visual inspection of their histograms, normal Q-Q plots

and box plots showed that the topical ointment and duration of diabetes mellitus (in years), CBG

on 1st visit, Hba1c (%), plasma albumin (gm%), duration of healed (in weeks), area of the ulcer,

duration of ulcer (in weeks), area of the ulcer at different follow up time period and Percentage

reduction in ulcer at different follow up time period parameters were non-normally distributed.67-

69

The comparison between topical ointment and duration of diabetes mellitus (in years), CBG on

1st visit, Hba1c (%), plasma albumin (gm%), duration of healed (in weeks), area of the ulcer,

duration of ulcer (in weeks), area of the ulcer at different follow up time period and Percentage

reduction in ulcer at different follow up time period was assessed by comparing the median

values. Mann Whitney U test was used to assess statistical significance. Data was also

represented using appropriate diagrams like box plots.

P value < 0.05 was considered statistically significant. IBM SPSS version 22 was used for

statistical analysis. 70

46

OBSERVATIONS AND RESULTS

47

RESULTS:RESULT:

A total 70 people were included in the analysis.

Table 1: Descriptive analysis of topical ointment in study population (N=70)

Topical ointment Frequency PercentagesPlacental Extract 35 50.00%Ionic Silver 35 50.00%

Among the study population, 35 (50%) people were placental extract and remaining 35 (50%)

people were ionic silver. (Table 1)

Table 2: Comparison of mean age (years) between study groups (N=70)

Topical ointment  AgeMean± STD

Mean difference

95% CIP value

Lower UpperPlacental Extract 59.74 ± 10.21

1.60 -3.47 6.67 0.531Ionic Silver 61.34 ± 11.05

The mean age of placental extract was 59.74 ± 10.21 and ionic silver was 61.34 ± 11.05, and the

mean difference (1.60) between two groups was statistically not significant (P value 0.531).

(Table 2 & Figure1)

Figure 1: Error bar diagram of comparison of mean age between study groups (N=70)

48

Table 3: Comparison of topical ointment with gender of study population (N=70)

GenderTopical ointment

Chi square P-valuePlacental Extract

(N=35)Ionic Silver

(N=35)Male 15 (42.9%) 26 (74.3%)

7.124 0.008Female 20 (57.1%) 9 (25.7%)

In placental extract group 15 (42.9%) people were male and remaining 20 (57.1%) people were

female. In ionic silver group 26 (74.3%) people were male and remaining 9 (25.7%) people were

female. The difference in the proportion of gender between topical ointment groups was

statistically significant (P value 0.008). (Table3 & Figure 2)

Figure 2: Cluster bar diagram of comparison of topical ointment with gender of study population (N=70)

49

Placental Extract Ionic Silver0.00%

10.00%

20.00%

30.00%

40.00%

50.00%

60.00%

70.00%

80.00%

42.90%

74.30%

57.10%

25.70%

Male Female

Topical ointment

Perc

enta

ge

Table 4: Comparison of median value of duration of diabetes mellitus (in years) between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Duration of diabetes

mellitus (in years)10 (7 to 18) 12 (8 to 20) 0.845

Among the people with placental extract, the median duration of diabetes mellitus (in years) was

10 years (IQR 7 to 18) and it was 12 years (IQR 8 to 20) in people with ionic silver. In

differencf.;e in the diabetes mellitus (in years) between topical ointment groups was statistically

not significant (p value 0.845). (Table 4 & Figure 3)

Figure 3: Box plots of comparison of median value of duration of diabetes mellitus (in years) between the two groups (N=70)

50

Table 5: Comparison of median value of CBG (mg/dl) on 1st visit between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

CBG on 1st visit 211 (135 to 322) 230 (138 to 294) 0.778

Among the people with placental extract, the median CBG on 1st visit was 211 (IQR 135 to 322)

and it was 230 (IQR 138 to 294) in people with ionic silver. In difference in the CBG on 1st visit

between topical ointment groups was statistically not significant (p value 0.778). (Table 5 &

Figure 4)

Figure 4: Box plots of comparison of median value of CBG on 1st visit between the two groups (N=70)

51

Table 6: Comparison of median value of Hba1c % between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Hba1c (%) 9.50 (8.10 to 12.50) 9.80 (8.50 to 12) 0.778

Among the people with placental extract, the median Hba1c (%) was 9.50 (IQR 8.10 to 12.50)

and it was 9.80 (IQR 8.50 to 12) in people with ionic silver. In difference in the Hba1c (%)

between topical ointment groups was statistically not significant (p value 0.778). (Table 6 &

Figure 5)

Figure 5: Box plots of comparison of median value of Hba1c % between the two groups (N=70)

52

Table 7: Comparison of Median value of plasma albumin (gm/dl) between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Plasma albumin (gm%) 3.60 (3.40 to 3.90) 3.80 (3.60 to 4) 0.164

Among the people with placental extract, the median plasma albumin (gm %) was 3.60 (IQR

3.40 to 3.90) and it was 3.80 (IQR 3.60 to 4) in people with ionic silver. In difference in the

plasma albumin (gm %) between topical ointment groups was statistically not significant (p

value 0.164). (Table 7& Figure 6)

Figure 6: Box plots of comparison of median value of Plasma albumin gm% between the two groups (N=70)

53

Table 8: Comparison of topical ointment with history of smoking alcohol intake in study population (N=70)

BehaviorTopical ointment

Chi square P-valuePlacental Extract

(N=35)Ionic Silver

(N=35)History of smoking

Yes 7 (20%) 8 (22.9%)0.085 0.771

No 28 (80%) 27 (77.1%)History of alcohol in take

Yes 7 (20%) 10 (28.6%)0.699 0.403

No 28 (80%) 25 (71.4%)

In placental extract group 7 (20%) people had smoking. In ionic silver group 8 (22.9%) people

had alcohol. The difference in the proportion of history of behaviours between topical ointments

groups was statistically not significant (P value 0.403). (Table 8)

Table 9: Comparison of topical ointment with pus at wound site of study population (N=70)

pus at wound site

Topical ointment Chi square P-valuePlacental Extract Ionic Silver

54

(N=35) (N=35)Yes 8 (22.9%) 13 (37.1%)

1.701 0.192No 27 (77.1%) 22 (62.9%)

In placental extract group 8 (22.9%) people had pus at wound site. In ionic silver group 13

(37.1%) people had pus at wound site. The difference in the proportion of pus at wound site

between topical ointments groups was statistically not significant (P value 0.192). (Table 9)

Table 10: Comparison of need for sloughectomy in study population (N=70)

SloughectomyTopical ointment Chi square P-valuePlacental Extract

(N=35)Ionic Silver

(N=35)Surgical 22 (62.9%) 28 (80%)

2.520 0.112No 13 (37.1%) 7 (20%)

In placental extract group 22 (62.9%) people had surgical. In ionic silver group 28 (80%) people

had surgical. The difference in the proportion of sloughectomy between topical ointments groups

was statistically not significant (P value 0.112). (Table 10)

Table 11: Comparison of topical ointment with swab culture and sensitivity of study population (N=70)

Swab culture and sensitivity

Topical ointmentChi square P-valuePlacental Extract

(N=35)Ionic Silver

(N=35)Staphylococcus aureus 5 (14.3%) 7 (20%) ** *

55

Klebsiella pneumonia 3 (8.6%) 2 (5.7%)Klebsiella oxytocin 1 (2.9%) 2 (5.7%)

Escherichia coli 2 (5.7%) 2 (5.7%)Citrobacter species 0 (0%) 1 (2.9%)

Pseudomonas aeruginosa 1 (2.9%) 3 (8.6%)Methicillin resistant

staphylococcus aureus 2 (5.7%) 2 (5.7%)

Proteus mirabilis 1 (2.9%) 1 (2.9%)Negative 20 (57.1%) 15 (42.9%)

**Chi square test not applicable. *No statistical test was applied- due to 0 subjects in the cells.

In placental extract group 5 (14.3%) participants had staphylococcus aureus, 3 (8.6%)

participants had klebsiella pneumonia, 1 (2.9%) participant had klebsiella oxytocin, 2 (5.7%)

participants had escherichia coli, 1 (2.9%) participant had pseudomonas aeruginosa, 2 (5.7%)

participants had methicillin resistant staphylococcus aureus, 1 (2.9%) participant had proteus

mirabilis. In ionic silver group 7 (20%) participants had staphylococcus aureus, 2 (5.7%)

participants had klebsiella pneumonia, 2 (5.7%) participants had klebsiella oxytocin, 2 (5.7%)

participants had escherichia coli, 1 (2.9%) participants had citrobacter species, 3 (8.6%)

participant had pseudomonas aeruginosa, 2 (5.7%) participants had methicillin resistant

staphylococcus aureus, 1 (2.9%) participant had proteus mirabilis. (Table 11)

Table 12: Comparison of topical ointment with healed cases in study population (N=70)

Healed casesTopical ointment

Chi square P-value

Placental Extract Ionic Silver

56

(N=35) (N=35)Yes 28 (80%) 27 (77.1%)

0.085 0.771No 7 (20%) 8 (22.9%)

In placental extract group 28 (80%) participants had healed cases. In ionic silver group 27

(77.1%) people had healed cases. The difference in the proportion of healed cases between

topical ointments groups was statistically not significant (P value 0.771). (Table 12 & figure 7)

Figure 7: Cluster bar chart comparison of topical ointment with healed cases in study population (N=70)

Placental Extract Ionic Silver0%

10%20%30%40%50%60%70%80%90% 80.00% 77.10%

20.00% 22.90%

Healed cases Yes Healed cases No

Topical ointment

Perc

enta

ge

Table 13: Comparison of Median taken for healing of chronic diabetic wounds (in weeks) between the two groups (N=70)

ParameterTopical ointment Mann Whitney

U test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Time taken for healed (in weeks)

6 (4 to 11) 8 (6 to 12) 0.030

57

Among the people with placental extract, the median duration of healed (in weeks) was 6 weeks

(IQR 4 to 11) and it was 8 weeks (IQR 6 to 12) in people with ionic silver. In difference in the

duration of healed (in weeks) between topical ointment groups was statistically significant (p

value 0.030). (Table 13 & figure 8)

Figure 8: Box plots of comparison of median taken for healing of chronic diabetic wounds (in weeks) between the two groups (N=70)

Table 14: Comparison of Median value of area of the ulcer between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Area of the ulcer 8 (3 to 24) 18 (6 to 32) 0.160

Among the people with placental extract, the median area of the ulcer was 8 (IQR 3 to 24) and it

was 18 (IQR 6 to 32) in people with ionic silver. In difference in the area of the ulcer between

topical ointment groups was statistically not significant (p value 0.160). (Table 14 Figure 9)

58

Figure 9: Box plots of comparison of median value of area of the ulcer between the two groups (N=70)

Table 15: Comparison of Median value of duration of ulcer (in weeks) between the two groups (N=70)

ParameterTopical ointment Mann Whitney U

test (P value)Placental Extract Median(IQR)

Ionic Silver Median(IQR)

Duration of ulcer (in weeks)

6 (4 to 8) 5 (4 to 8) 0.951

Among the people with placental extract, the median duration of ulcer (in weeks) was 6 weeks

(IQR 4 to 8) and it was 5 weeks (IQR 4 to 8) in people with ionic silver. In difference in the

duration of ulcer (in weeks) between topical ointment groups was statistically not significant (p

value 0.951). (Table 15 & Figure 10)

Figure 10: Box plots of comparison of median value of duration of ulcer (in weeks) between the two groups (N=70)

59

Table 16: Comparison of mean time taken for compelete granulation (in days) between study groups (N=70)

Topical ointment Time taken for compel granulation (in days)

Mean± STD

Mean difference

95% CIP valueLower Upper

Placental Extract 13.56 ± 5.752.15 -0.00 4.30 0.050

Ionic Silver 15.71 ± 1.76

The mean time taken for compel granulation (in days) of placental extract was 13.56 ± 5.75 and

ionic silver was 15.71 ± 1.76, and the mean difference (2.15) between two groups was

statistically significant (P value 0.050). (Table 16 & Figure 11)

Figure 11: Error bar diagram of comparison of mean time taken for complete granulation

(in days) between study groups (N=70)

60

Table 17: Comparison of median area of the ulcer at different follow up time period in both the groups in Healed cases

Area of the ulcer at follow up

Topical ointmentMann Whitney U

test(P value)Placental Extract

Median(IQR)Ionic Silver

Median(IQR)End of 1st week 6.40 (2.16 to 21.12) 13.05 (4.26 to 26.24) 0.198End of 4th week 2.16 (0.32 to 9.60) 6.15 (1.80 to 15) 0.058End of 6th week 0.00 (0.00 to 4.50) 3.60 (0.00 to 10.00) 0.073End of 9th week 0.00 (0.00 to 3.00) 0.00 (0.00 to 3.20) 0.499End of 12th week 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.308

There is no statistical difference in area of the ulcer at end of 1 st week, 4th week, 6th weeks, 9th

week and 12th week between topical ointment groups (>0.05). (Table 17)

Table 18: Comparison of median percentage reduction in ulcer at different follow up time period in both the groups in Healed cases

Percentage reduction in ulcer

Topical ointment Mann Whitney U test

(P value)Placental Extract

Median(IQR)Ionic Silver

Median(IQR)

61

End of 1st week 20 (13 to 34) 20 (15 to 26) 0.842End of 4th week 65 (60 to 92) 64 (55 to 74) 0.166End of 6th week 100 (76 to 100 ) 85 (76 to 100) 0.230End of 9th week 100 (88.40 to 100) 100 (90 to 100) 0.702End of 12th week 100 (100 to 100) 100 (100 to 100) 0.327

There is no statistical difference in percentage reduction in ulcer at end of 1st week, 4th weeks, 6th

weeks, 9th week and 12th week between topical ointment groups (>0.05). (Table 18)

Table 19: Comparison of median ulcer remaining at different follow up time period in both the groups in Healed cases

Ulcer remainingTopical ointment Mann Whitney U

test(P value)

Placental Extract Median(IQR)

Ionic Silver Median(IQR)

End of 1st week 79 (65.75 to 84.75) 80 (74 to 85) 0.692End of 4th week 31 (0.00 to 40) 36 (26 to 45) 0.048End of 6th week 0.00 (0.00 to 16.25) 10 (0.00 to 24) 0.047End of 9th week 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.567End of 12th week 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 1.00

There is no statistical difference in ulcer remaining at end of 1st week, 9th week and 12th week

between topical ointment groups (>0.05). Among the people with placental extract group, the

median ulcer remaining at end of 4th week was 31 (IQR 0.00 to 40) and it was 36 (IQR 26 to 45)

in people with ionic silver. The difference in the ulcer remaining at end of 4th week between

topical ointment groups was statistically significant. (P value 0.048). Among the people with

placental extract group, the median ulcer remaining at end of 6th week was 0.00 (IQR 0.00 to

16.25) and it was 10 (IQR 0.00 to 24) in people with ionic silver. The difference in the ulcer

remaining at end of 6th week between topical ointment groups was statistically significant. (P

value 0.047). (Table 19)

62

63

DISCUSSION

DISCUSSION:

64

Diabetes mellitus (DM) is a chronic disorder of glucose metabolism with multi-organ system

involvement and serious long term consequences. Prevalence of diabetes in India is around

8%.71Diabetes is increasing in India and other countries due to changes in food habits and

lifestyle similar to western style. And better health service and facility available but more

patients come with chronic complication. Diabetics are predisposed to development of foot

lesions, owing to a complex association of various factors like ischemia, neuropathy and

infection. Micro-angiopathy, hypercoagulable stat, atherosclerosis, and hyperglycemia may leads

the above complex with eventual diabetes lesions and complications.9, 10, 72

Foot disorders such as ulceration, infection, and gangrene are the leading causes of

hospitalization in patients with diabetes mellitus. The diabetic foot and its sequelae account for

billions of dollars in direct medical expenditures, as well as lengthy hospital stays and periods of

disability.9, 10

Foot ulceration is thought to affect 15% of people with diabetes at some time in their

lives.73 People with diabetes are between 15 and 70 times more likely to undergo lower limb

amputations than people without diabetes.74 In 2002, the age- adjusted lower extremity

amputation rate among men was 7.0 per 1,000 persons with diabetes compared with the rate

among women reported as 3.3 per 1000 persons with diabetes.75

Although numerous topical medications and gels are promoted for ulcer care, relatively

few have proved to be more efficacious than saline wet-to-dry dressings.76, 77 Generally, a

warm, moist environment that is protected from external contamination is most conducive to

wound healing. This can be provided by a number of commercially available special dressings.

65

Adequate debridement must always precede the application of topical wound healing

agents, dressings or wound closure procedures.78 Elderly diabetics are usually unable to notice

the foot lesions due to poor sight and are also unable to feel duet to sensory loss, there by

predisposing them to diabetic foot. The common precipitating factors are mechanical, thermal,

chemical or surgical wound.79

Among the study population, 35 (50%) people were placental extract and remaining 35

(50%) people were ionic silver. The mean age of the patients receiving placental extract was

59.74 ± 10.21 years and those with ionic silver was 61.34 ± 11.05 years. A slightly lower but still

elderly patients were investigated by Vivek Vardhan and Nagateja80 with an average age of

patients Placental extract group being 55.7 years and that in the conventional group it was 57.9

years and by Navadiya et al.81 (57 years in Group A and 55 years in Group B).

The gender distribution showed females outnumber males in placental group (57.1% vs.

42.9%), while in the ionic silver group, considerable male preponderance was noted (74.3% vs

25.7%). A significant difference (0.008) in the gender difference was noted. In their

investigation of 100 Indian diabetic foot ulcer patients, Vivek Vardhan and Nagateja80 noted a

significantly higher male patients in both placental group (70% vs 30%) and the conventional

group (74% vs 26%).

Vivek Vardhan and Nagateja80 reported their patients with duration a diabetes of about

6.8 years in the group P and in the group C it was 5.6 years, while Navadiya et al.81 noted 6.1

years in group A and 7.7 years in group B. Comparatively our study cases had a longer duration

of diabetes among both groups, where in the median duration was 10 years (IQR 7 to 18) in the

placental extract group, while it was 12 years (IQR 8 to 20) in ionic silver group.

66

Among the people with placental extract, the median CBG on 1st visit was 211 (IQR 135

to 322) and it was 230 (IQR 138 to 294) in people with ionic silver.

Among the people with placental extract, the median HbA1c (%) was 9.50 (IQR 8.10 to

12.50) and it was 9.80 (IQR 8.50 to 12) in people with ionic silver. However the HbA1c values

were slightly less among the patients studied by Vivek Vardhan and Nagateja80 (Group C, 8.4%

and in Group P, 8.3%) and was higher in the study by Aziz et al. (15% of the patients had

HbA1c>13.0%).

Among the people with placental extract, the median plasma albumin (gm %) was 3.60 (IQR

3.40 to 3.90) and it was 3.80 (IQR 3..60 to 4) in people with ionic silver.

About 7 (20%) patients were smokers in placental extract group 7 (20%) while in ionic silver

group 8 (22.9%) people had alcohol.

In placental extract group 8 (22.9%) people had pus at wound site. In ionic silver group 13

(37.1%) people had pus at wound site.

Sloughectomy In placental extract group 22 (62.9%) people had surgical. In ionic silver group 28

(80%) people had surgical.

In placental extract group 5 (14.3%) participants had Staphylococcus aureus, 3 (8.6%)

participants had Klebsiella pneumonia, In ionic silver group 7 (20%) participants had

Staphylococcus aureus, 3 (8.6%) participant had Pseudomonas aeruginosa. A prospective study

by Aziz et al.75 investigating the predictive factors for lower extremity amputations in diabetic

67

foot infections found that Staphylococcus aureus as the commonest microbe (39.7%) in both

monomicrobial and polymicrobial infections followed by Bacteroides fragilis (30.3%) and

Pseudomonas aeruginosa (26.0%).

Patients who have been previously hospitalized with an open wound are more likely to

develop an infection from resistant bacteria such as methicillin-resistant S aureus (MRSA) and

vancomycin-resistant enterococci (VRE).82 Chronic wounds can develop a more complex

assortment of bacteria, including gram-negative rods, obligate anaerobes, Pseudomonas

aeruginosa, and enterococci. The key pathogens are more reliably detected in specimens that are

obtained deep rather than superficial swabs. The IDSA (Infectious Disease Society of America)

formulated guidelines and key recommendations for treatment of DFU stating that an empirical

antibiotic regimen should be implemented primarily on the basis of infection severity and likely

pathologic agents. Optimally, definitive therapy should be based upon culture and susceptibility

analysis.

In placental extract group 28 (80%) participants had healed cases. In ionic silver group 27

(77.1%) people had healed cases. The difference in the proportion of healed cases between

topical ointments groups was statistically not significant (P value 0.771). Treatment silver nano

particles as compared to conventional dressing has been found to be cost effective and

considerably reduces the morbidity related to amputation in diabetes.62, 83 More than 10 different

silver-containing dressings, including silver-containing hydrogels, hydrofibers, and alginates, are

currently available worldwide. Although all are assumed to be safe and effective, evidence of

their efficacy is scant; few clinical trials have been performed with them.84 A recent systematic

review and meta-analysis by Carter et al.12 concluded that though there is some evidence that

68

silver-impregnated dressings improve the short-term healing of wounds and ulcers, long-term

effects remain unclear.

The ulcers treated with placental extract healed significantly (P=0.03) in shorter duration

(6 weeks) compared to those treated with ionic silver (8 weeks). In line with these findings,

Navadiya et al.81 observed that diabetic foot ulcers treated with povidone Iodine required 8.3

more days to heal compared to those with placental extract.

Among the people with placental extract, the median area of the ulcer was 8 (IQR 3 to 24) and it

was 18 (IQR 6 to 32) in people with ionic silver.

The mean time taken for complete granulation of placental extract was 13.56 ± 5.75 days was

significantly lower than that of ionic silver (15.71 ± 1.76 days).

There is no statistical difference in area of the ulcer at end of 1 st week, 4th week, 6th weeks, 9th

week and 12th week between topical ointment groups (>0.05).

Among the people with placental extract group, the median ulcer remaining at end of 4th

week was 31 (IQR 0.00 to 40) and it was 36 (IQR 26 to 45) in people with ionic silver. The

difference in the ulcer remaining at end of 4th week between topical ointment groups was

statistically significant. (P value 0.048). Among the people with placental extract group, the

median ulcer remaining at end of 6th week was 0.00 (IQR 0.00 to 16.25) and it was 10 (IQR 0.00

to 24) in people with ionic silver. The difference in the ulcer remaining at end of 6th week

between topical ointment groups was statistically significant. (P value 0.047). Similar findings

69

of placental extract treatment requiring lesser duration of time and faster healing was reported by

Navadiya et al.81 (1-20 days: 20% in placental extract group and 13.4% in povidone Iodine

group; 21-40 days: 73.3% in group A and 59.9% in group B) with all the wounds in group A

completely healed by 60 days while 6.7% of them still had incomplete wound healing in group

B. Mounting evidence to suggest that diabetic wounds treated with topical placental extracts had

lesser hospital stay, lesser overall treatment time and better wound healing.80, 81

Placental extract increases collagen synthesis, increases tissue protein, accelerates

neoangiogenesis, and epithelialization. It having immunotropic effect and having EGF,

Fibroblast growth factor. It supports ossification and reduces surrounding tissue inflammation

and edema.45, 47, 48

Figure 1: Cellular Mechanism of repair in Placental Therapy

Placental extracts can be classified into two different types: aqueous extract and

hydroalcoholic extract. The components present in the extract depend on the method of its

70

preparation and are based on solubility of the components in respective solvent of extraction.

Thus, an aqueous extract is likely to contain more polar molecules such as peptides/proteins,

small organic components like amino acids, nucleotides, polydeoxyribonucleotides (PDRNs),

carbohydrates and trace amount of lipids mostly bound to proteins which are comparatively

soluble in aqueous medium. Likewise, various types of lipids may be present in hydroalcoholic

extract (less polar and hydrophobic). Chemical analysis of the hydroalcoholic extract revealed

the presence of glycosphingolipids, cholesterol, triglycerides, high density lipoproteins,

carbohydrates, sialic acids and others, including amino acids, nucleotides, carotenes, vitamins,

including small amount of low-molecular-weight proteins/peptides containing hydrophobic

amino acid residues which are soluble in a less polar solvent. Modern indigenous aqueous

placental extract is prepared employing Filatov’s procedure.

Human placental extract manufactured by a proprietary extraction method using term

human placenta as a raw material has potent therapeutic efficacy. Use of placental extract in

different preparations under various trade names is a recommended worldwide practice.

Developed from folk knowledge, the aqueous extract is used as a globally accepted licensed drug

in post surgical dressings, as a healer in burn injuries and chronic wounds under different trade

name in many countries including India (Chakraborty et al., 2009).

Chakraborty et al. studied the role of placenta on the growth of different bacteria. They

found that placenta prevents the growth of bacteria such as E. coli from urine and blood culture.

They found placenta to also have an inhibitory role in the growth of bacteria such as E. coli,

Staphylococcus aureus and fungi such as Saccharomyces cerevisae, Kluyvero-myces fragilis, and

Candida albicans.

71

Decreased hospital stay and treatment time reduces the burden on hospital as well as

early recovery on the part of patient. Other important thing is required to concentrate more on

preventive measure and patient's self-care in diabetic patient.

Treatment of the underlying ischemia is critical in achieving a successful outcome,

regardless of topical therapies. Patient education has a central role in treatment and should

include instruction on foot hygiene, daily inspection, proper footwear, and the Antibiotic

coverage should subsequently be tailored according to the clinical response of the patient, culture

results, and sensitivity testing. Surgical drainage, deep debridement, or local partial foot

amputations are necessary adjuncts to antibiotic therapy of infections. Regular foot-care

examinations, including debridement of calluses and ingrown toe-nails, provide an opportunity

to reinforce appropriate self-care behaviors and allow for early detection of new or impending

foot problems.85

Conclusion

The study findings suggest that diabetic foot ulcers treated with topical placental extracts was as

equally effective as ionic silver significantly reducing the duration of healing. There were no

adverse effects or reactions observed in any of the patients with the placental extract. Natural

medicine continues to play an important role for prevention, alleviation and cure of diseases. In

some part of the Western world, the use of traditional medicine has been largely lost. However, it

is a widespread phenomenon in the developing countries where 80% of the population is still

relying on traditional medicine for primary healthcare. Derived from folklore, human placental

72

preparations show immense therapeutic value and can be safely.

Diabetes is usually a lifelong disease. As the healthcare facility is gradually improving

more and more patients are presenting with chronic complications of diabetes like neuropathy,

foot lesion, and retinopathy. Diabetic foot is the most common complication of diabetic patient

and is not totally avoidable but with positive approach morbidity and mortality due to diabetic

foot problems can definitely be reduced. In operative patient history of diabetes requires a

special care, even after that there are higher rate of post operative complication such as wound

infection compare to non-diabetics. Higher tissue glucose level is mostly responsible for all

events. Very few studies have been done to test the efficacy of purified placental extract on foot

ulcers; needs studies with larger study groups to test efficacy of placental extract on various

wounds.

73

BIBLIOGRAPHY

REFERENCE:

1. Whiting DR, Guariguata L, Weil C, Shaw J. IDF diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. Diabetes research and clinical practice. 2011;94(3):311-21.2. Cho NH, Shaw JE, Karuranga S, Huang Y, da Rocha Fernandes JD, Ohlrogge AW, et al. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pract. 2018;138:271-81.3. Anjana RM, Deepa M, Pradeepa R, Mahanta J, Narain K, Das HK, et al. Prevalence of diabetes and prediabetes in 15 states of India: results from the ICMR-INDIAB population-based cross-sectional study. Lancet Diabetes Endocrinol. 2017;5(8):585-96.4. organization WH. Global Health Risks-Mortality and burden of disease attributable to selected major risks. Cancer. 2017.

74

5. Mohan V, Shah S, Saboo B. Current glycemic status and diabetes related complications among type 2 diabetes patients in India: data from the A1chieve study. J Assoc Physicians India. 2013;61(1 Suppl):12-5.6. Zhang P, Lu J, Jing Y, Tang S, Zhu D, Bi Y. Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis (dagger). Ann Med. 2017;49(2):106-16.7. Ragnarson Tennvall G, Apelqvist J. Health-economic consequences of diabetic foot lesions. Clinical Infectious Diseases. 2004;39(Supplement_2):S132-S9.8. Rice JB, Desai U, Cummings AKG, Birnbaum HG, Skornicki M, Parsons NB. Burden of diabetic foot ulcers for medicare and private insurers. Diabetes care. 2014;37(3):651-8.9. Alavi A, Sibbald RG, Mayer D, Goodman L, Botros M, Armstrong DG, et al. Diabetic foot ulcers: Part I. Pathophysiology and prevention. J Am Acad Dermatol. 2014;70(1):1.e-18; quiz 9-20.10. Damir A. Why Diabetic Foot Ulcers do not heal? J International Medical Sciences Academy. 2011;24(4):205-6.11. Chakraborty PD, Bhattacharyya D. Isolation of fibronectin type III like peptide from human placental extract used as wound healer. Journal of Chromatography B. 2005;818(1):67-73.12. Carter MJ, Tingley-Kelley K, Warriner RA, 3rd. Silver treatments and silver-impregnated dressings for the healing of leg wounds and ulcers: a systematic review and meta-analysis. J Am Acad Dermatol. 2010;63(4):668-79.13. Chaw K, Manimaran M, Tay FE. Role of silver ions in destabilization of intermolecular adhesion forces measured by atomic force microscopy in Staphylococcus epidermidis biofilms. Antimicrobial agents and chemotherapy. 2005;49(12):4853-9.14. Gan D. Diabetes atlas: International Diabetes Federation; 2003.15. Atlas D. International diabetes federation. Press Release, Cape Town, South Africa. 2006;4.16. Guariguata L, Whiting DR, Hambleton I, Beagley J, Linnenkamp U, Shaw JE. Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes research and clinical practice. 2014;103(2):137-49.17. Ogurtsova K, da Rocha Fernandes J, Huang Y, Linnenkamp U, Guariguata L, Cho N, et al. IDF Diabetes Atlas: Global estimates for the prevalence of diabetes for 2015 and 2040. Diabetes research and clinical practice. 2017;128:40-50.18. Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes care. 2004;27(5):1047-53.19. Ahuja MMS. Epidemiology of diabetes in developing countries: Interprint; 1979.20. Sridhar G, Rao P, Ahuja M. Epidemiology of diabetes and its complications. RSSDI textbook of diabetes mellitus Hyderabad: Research Society for the Study of Diabetes in India. 2002;1:95-112.21. Rao P, Ushabala P, Seshiah V, Ahuja M, Mather H. The Eluru survey: prevalence of known diabetes in a rural Indian population. Diabetes research and clinical practice. 1989;7(1):29-31.22. Kolluru GK, Bir SC, Kevil CG. Endothelial dysfunction and diabetes: effects on angiogenesis, vascular remodeling, and wound healing. International journal of vascular medicine. 2012;2012.23. Brownlee M. The pathobiology of diabetic complications: a unifying mechanism. diabetes. 2005;54(6):1615-25.24. Forbes JM, Cooper ME. Mechanisms of diabetic complications. Physiological reviews. 2013;93(1):137-88.25. Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global burden of diabetic foot disease. The Lancet. 2005;366(9498):1719-24.26. Raghav A, Khan ZA, Labala RK, Ahmad J, Noor S, Mishra BK. Financial burden of diabetic foot ulcers to world: a progressive topic to discuss always. Ther Adv Endocrinol Metab. 2018;9(1):29-31.27. Ghosh P, Valia R. Burden of Diabetic Foot Ulcers in India: Evidence Landscape from Published Literature. Value in Health. 2017;20(9):A485.

75

28. Geerlings SE, Hoepelman AI. Immune dysfunction in patients with diabetes mellitus (DM). Pathogens and Disease. 1999;26(3 4):259-65.‐29. Delamaire M, Maugendre D, Moreno M, Le Goff MC, Allannic H, Genetet B. Impaired leucocyte functions in diabetic patients. Diabetic Medicine. 1997;14(1):29-34.30. Mowat AG, Baum J. Chemotaxis of polymorphonuclear leukocytes from patients with diabetes mellitus. New England journal of medicine. 1971;284(12):621-7.31. Tater D, Tepaut B, Bercovici J, Youinou P. Polymorphonuclear cell derangements in type I diabetes. Hormone and metabolic research. 1987;19(12):642-7.32. Marhoffer W, Stein M, Maeser E, Federlin K. Impairment of polymorphonuclear leukocyte function and metabolic control of diabetes. Diabetes care. 1992;15(2):256-60.33. Boulton AJ. Management of diabetic peripheral neuropathy. Clinical diabetes. 2005;23(1):9-15.34. Mustoe T. Understanding chronic wounds: a unifying hypothesis on their pathogenesis and implications for therapy. The American Journal of Surgery. 2004;187(5):S65-S70.35. Fowler MJ. Microvascular and macrovascular complications of diabetes. Clinical diabetes. 2008;26(2):77-82.36. Calles-Escandon J, Cipolla M. Diabetes and endothelial dysfunction: a clinical perspective. Endocrine reviews. 2001;22(1):36-52.37. Crovetti G, Martinelli G, Issi M, Barone M, Guizzardi M, Campanati B, et al. Platelet gel for healing cutaneous chronic wounds. Transfusion and Apheresis Science. 2004;30(2):145-51.38. Watters C, Trivedi U, Deleon K, Dalton T, Rumbaugh KP, editors. Bacterial Biofilms Contribute to the Chronicity and Antimicrobial Resistance of Diabetic Chronic Wound Infections. DIABETES; 2010: AMER DIABETES ASSOC 1701 N BEAUREGARD ST, ALEXANDRIA, VA 22311-1717 USA.39. Banu A, Hassan MMN, Rajkumar J, Srinivasa S. Spectrum of bacteria associated with diabetic foot ulcer and biofilm formation: a prospective study. The Australasian medical journal. 2015;8(9):280.40. Goldstein S, Moerman E, Soeldner J, Gleason R, Barnett D. Diabetes mellitus and genetic prediabetes. Decreased replicative capacity of cultured skin fibroblasts. The Journal of clinical investigation. 1979;63(3):358-70.41. Loots MA, Lamme EN, Mekkes JR, Bos JD, Middelkoop E. Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation. Archives of dermatological research. 1999;291(2-3):93-9.42. Augustin M, Maier K. Psychosomatic aspects of chronic wounds. Dermatology and Psychosomatics/Dermatologie und Psychosomatik. 2003;4(1):5-13.43. Chase SK, Melloni M, Savage A. A forever healing: the lived experience of venous ulcer disease. Journal of vascular nursing. 1997;15(2):73-8.44. Ismail K, Winkley K, Stahl D, Chalder T, Edmonds M. A cohort study of people with diabetes and their first foot ulcer: the role of depression on mortality. Diabetes care. 2007;30(6):1473-9.45. Fang XP, Xia WS, Sheng QH, Wang YL. Purification and Characterization of an Immunomodulatory Peptide from Bovine Placenta Water Soluble Extract. Preparative biochemistry &‐ biotechnology. 2007;37(3):173-84.46. Chakraborty P, De D, Bandyopadhyay S, Bhattacharyya D. Human aqueous placental extract as a wound healer. Journal of wound care. 2009;18(11):462-7.47. O'keefe E, Payne R, Russell N. Keratinocyte growth promoting activity from human placenta.‐ Journal of cellular physiology. 1985;124(3):439-45.48. Carotti D, Allegra E. An approach to chemical characterization of human placental extracts: proteins, peptides, and amino acids analyses. Physiological chemistry and physics. 1981;13(2):129-36.49. Woo K, Ayello E, Sibbald R. SILVER versus other antimicrobial dressings: best practices! Surgical technology international. 2008;17:50-71.

76

50. Lansdown AB. Silver I: its antibacterial properties and mechanism of action. Journal of wound care. 2002;11(4):125-30.51. Parsons D, Bowler PG, Myles V, Jones S. Silver antimicrobial dressings in wound management: a comparison of antibacterial, physical, and chemical characteristics. Wounds-a compendium of clinical research and practice. 2005;17(8):222-32.52. Wilkinson L, White R, Chipman J. Silver and nanoparticles of silver in wound dressings: a review of efficacy and safety. Journal of wound care. 2011;20(11):543-9.53. Cutting K, White R, Edmonds M. The safety and efficacy of dressings with silver–addressing clinical concerns. International wound journal. 2007;4(2):177-84.54. Leaper D. Appropriate use of silver dressings in wounds: international consensus document. International wound journal. 2012;9(5):461-4.55. Lansdown AB. A pharmacological and toxicological profile of silver as an antimicrobial agent in medical devices. Advances in pharmacological sciences. 2010;2010.56. Leaper DJ. Silver dressings: their role in wound management. International wound journal. 2006;3(4):282-94.57. Failla CM, Odorisio T, Cianfarani F, Schietroma C, Puddu P, Zambruno G. Placenta growth factor is induced in human keratinocytes during wound healing. J Invest Dermatol. 2000;115(3):388-95.58. Chauhan VS, Rasheed MA, Pandley SS, Shukla VK. Nonhealing wounds--a therapeutic dilemma. Int J Low Extrem Wounds. 2003;2(1):40-5.59. Coutts P, Sibbald RG. The effect of a silver-containing Hydrofiber dressing on superficial wound bed and bacterial balance of chronic wounds. Int Wound J. 2005;2(4):348-56.60. Jorgensen B, Price P, Andersen KE, Gottrup F, Bech-Thomsen N, Scanlon E, et al. The silver-releasing foam dressing, Contreet Foam, promotes faster healing of critically colonised venous leg ulcers: a randomised, controlled trial. Int Wound J. 2005;2(1):64-73.61. Cianfarani F, Zambruno G, Brogelli L, Sera F, Lacal PM, Pesce M, et al. Placenta growth factor in diabetic wound healing: altered expression and therapeutic potential. Am J Pathol. 2006;169(4):1167-82.62. Bergin SM, Wraight P. Silver based wound dressings and topical agents for treating diabetic foot ulcers. Cochrane Database Syst Rev. 2006(1):Cd005082.63. Jude EB, Apelqvist J, Spraul M, Martini J. Prospective randomized controlled study of Hydrofiber dressing containing ionic silver or calcium alginate dressings in non-ischaemic diabetic foot ulcers. Diabet Med. 2007;24(3):280-8.64. Vermeulen H, van Hattem JM, Storm-Versloot MN, Ubbink DT. Topical silver for treating infected wounds. Cochrane Database Syst Rev. 2007(1):Cd005486.65. Shapiro SS, Wilk MB. An analysis of variance test for normality (complete samples). Biometrika. 1965;52(3/4):591-611.66. Razali NM, Wah YB. Power comparisons of shapiro-wilk, kolmogorov-smirnov, lilliefors and anderson-darling tests. Journal of statistical modeling and analytics. 2011;2(1):21-33.67. Cramer D. Fundamental statistics for social research: step-by-step calculations and computer techniques using SPSS for Windows: Psychology Press; 1998.68. Cramer D, Howitt DL. The Sage dictionary of statistics: a practical resource for students in the social sciences: Sage; 2004.69. Doane DP, Seward LE. Measuring skewness: a forgotten statistic? Journal of Statistics Education. 2011;19(2).70. MacGregor L, Day K. International consensus: Appropriate use of silver dressings in wounds. Wounds International Enterprise House. 2012.71. Ramachandran A, Das A, Joshi S, Yajnik C, Shah S, Kumar KP. Current status of diabetes in India and need for novel therapeutic agents. J Assoc Physicians India. 2010;58:7-9.

77

72. Levin ME. An overview of the diabetic foot: pathogenesis, management and prevention of lesions. Int J diab dev Countries. 1994;14:39-47.73. Mishra SC, Chhatbar KC, Kashikar A, Mehndiratta A. Diabetic foot. Bmj. 2017;359:j5064.74. Campbell IW, Nairn M. Scottish Intercollegiate Guidelines network: management of diabetes (SIGN 55). The British Journal of Diabetes & Vascular Disease. 2002;2(1):50-2.75. Aziz Z, Lin WK, Nather A, Huak CY. Predictive factors for lower extremity amputations in diabetic foot infections. Diabet Foot Ankle. 2011;2.76. O'Loughlin A, McIntosh C, Dinneen SF, O'Brien T. Review paper: basic concepts to novel therapies: a review of the diabetic foot. Int J Low Extrem Wounds. 2010;9(2):90-102.77. Sridharan K, Sivaramakrishnan G. Growth factors for diabetic foot ulcers: mixed treatment comparison analysis of randomized clinical trials. Br J Clin Pharmacol. 2018;84(3):434-44.78. Consensus Development Conference on Diabetic Foot Wound Care: 7-8 April 1999, Boston, Massachusetts. American Diabetes Association. Diabetes Care. 1999;22(8):1354-60.79. Singer AJ, Clark RA. Cutaneous wound healing. New England journal of medicine. 1999;341(10):738-46.80. Vivek VJ, Nagateja M. A comparative analysis of the merits of topical placental extract over conventional methods of dressings for diabetic foot ulcers. Journal of Evolution of Medical and Dental Sciences. 2017;6(12):963-7.81. Navadiya SK, Vaghani YL, Patel MP. Study of topical placental extract versus povidone iodine and saline dressing in various diabetic wounds. National J Med Res. 2012;2:411-3.82. Hartemann Heurtier A, Robert J, Jacqueminet S, Ha Van G, Golmard J, Jarlier V, et al. Diabetic‐ foot ulcer and multidrug resistant organisms: risk factors and impact. Diabetic Medicine.‐ 2004;21(7):710-5.83. Gupta BK, Jain S. A NON-RANDOMISED CONTROLLED TRIAL STUDY OF THE EFFECT OF NANO SILVER PARTICLES AND CONVENTIONAL DRESSING ON THE HEALING OF CHRONIC ULCER. Chest. 2017;1:01.84. Hermans MH. Silver-containing dressings and the need for evidence. Advances in skin & wound care. 2007;20(3):166-73.85. Frykberg RG. Diabetic foot ulcers: pathogenesis and management. Am Fam Physician. 2002;66(9):1655-62.

78

ANNEXURES

79