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TRANSCRIPT
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
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Glossary Abbreviations
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INTRODUCTION
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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.
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AIMS & OBJECTIVES
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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
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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
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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
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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
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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
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ANNEXURES
79