THE UNIVERSITY OF MEDICINE AND
PHARMACY GRIGORE T. POPA IAȘI
DETERMINANTS OF OSTEOARTICULAR FOOT
CHANGES IN PATIENTS WITH DIABETES
Scientific leader
Prof.Univ.Dr.GRAUR Mariana
PhD student:
NIȚĂ George
2021
Keywords: diabetic foot, bone turnover, RANKL, FGF23, survival.
The doctoral thesis includes: • 236 pages, of which 40 representing the general part
(current state of knowledge) - structured in 3 chapters and 196 representing the
personal part (own contribution) - structured in 4 chapters • 79 figures, 191 tables; •
4 annexes; • 451 bibliographical references.
The table of contents of the abstract is kept in the same form as in the doctoral
thesis.
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TABLE OF CONTENTS
LIST OF ABBREVIATIONS INTRODUCTION.............................................................................................................. 1
THE GENERAL PART
Chapter 1. Diabetes and the osteoarticular system ................................................... 2
1.1. Bone physiology and biology .................................................................................. 2
1.2. Markers of bone turnover ........................................................................................ 4
1.3. Mineral homeostasis and bone turnover in diabetes ............................................... 7
1.4. Skeletal changes in diabetes .................................................................................... 9
1.5. Implications of the RANK/RANKL/OPG system ................................................. 10
1.6. The role of FGF23 ................................................................................................. 11
Chapter 2. Diabetic foot ............................................................................................. 13
2.1. Epidemiology and economic implications of diabetic foot ................................. 13
2.2. Pathogenesis of diabetic foot ................................................................................ 15
2.3. Charcot osteoarthropathy .................................................................................... 17
2.4. Evaluation of the Charcot foot ............................................................................. 19
2.5. Charcot foot treatment .......................................................................................... 21
Chapter 3. Surgical diabetic foot .............................................................................. 24
3.1. The importance of the problem ............................................................................. 24
3.2. Classification of diabetic foot ulcers ..................................................................... 25
3.3. Risk factors for diabetic foot ................................................................................. 33
3.4. Risk factors for fractures ....................................................................................... 37
3.5. Factors influencing ulcer healing .......................................................................... 38
THE PERSONAL PART
Chapter 4. Retrospective study evaluating predictors of the evolution of diabetic foot
ulcers
4.1. Motivation and objectives of the doctoral study ............................................... 41
4.2. Material and methods ......................................................................................... 42
4.3. Results .................................................................................................................. 51
4.4. Discussions ......................................................................................................... 104
4.5. Conclusions ........................................................................................................ 130
Chapter 5. Observational analytical study to assess bone turnover in patients with
type 2 diabetes using modern biomarkers
5.1. Motivation and objectives of the doctoral study ............................................. 131
5.2. Material and methods ....................................................................................... 132
5.3. Results ................................................................................................................ 135
5.4. Discussions ......................................................................................................... 194
5.5. Conclusions ........................................................................................................ 209
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Chapter 6. General conclusions ................................................................................... 211
Chapter 7. Aspects of originality and perspectives opened by the thesis .................. 212
BIBLIOGRAPHY .......................................................................................................... 214
ANNEXES
ANNEX 1. Legend for retrospective study database
ANNEX 2. Observational study patient file
ANNEX 3. Food frequency questionnaire
ANNEX 4. Physical activity questionnaire
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Introduction. Current state of knowledge
Diabetes mellitus (DM) is one of the most common chronic non-communicable
diseases globally. The global burden of diabetes will increase from 463 million people
with diabetes in 2019 to 700 million in 2045 (51% increase) (1). DM is the fifth leading
cause of death in most countries, contributing to 1.5 million deaths annually (2).
DM has a major impact on metabolism, having many complications and
contributing to increased mortality through an increased risk of coronary heart disease and
stroke (1). Moreover, the complications of diabetes are often complex disorders, such as
diabetic foot syndrome, one of the major causes of non-traumatic lower limb amputation.
Diabetic foot is a dreaded complication of diabetes, with long-term hospitalizations and
significant costs, often the end being amputation. Patients with diabetic foot disease fear a
major amputation of the lower limbs more than death. The variables that were associated
with the classification of lower limb amputation as the greatest fear were the presence of a
leg complication related to diabetes, the duration of diabetes ≥ 10 years, insulin use and
the presence of peripheral neuropathy (3).
The diabetic foot is characterized by a classic triad of neuropathy, ischemia and
infection. It is currently an important public health issue, and data on its global
epidemiology as well as predictions for the future are extremely worrying. The first
priority should be to prevent diabetic foot. This can be achieved through good disease
control and early identification of high-risk individuals, such as those with peripheral
neuropathy, peripheral vascular disease, leg deformities, and the presence of callus.
Understanding the etiological and risk factors of diabetic foot ulcers can help improve
quality of life and reduce the complications of foot ulcers. Given the dramatic increase in
the incidence of diabetic foot infections, it is crucial to identify certain risk factors among
predefined predisposing factors. Identifying the most important risk factors among these
multiple variables can help determine diagnostic and treatment protocols when
considering disease management, setting treatment priorities, and patient quality of life.
The personal part
Chapter 4. Retrospective study evaluating predictors of the evolution
of diabetic foot ulcers
4.1. Motivation and objectives of the doctoral study
In recent years, DM and its complications have quickly become the most
significant cause of morbidity and mortality. DM is a challenge for the 21st century, as the
number of diabetics has more than tripled in the last 20 years. Thus, diabetic foot and
lower limb complications affect millions of diabetics globally, representing a huge source
of morbidity (1). It is estimated that worldwide, every 30 seconds, an amputation of the
lower limb is performed due to DM (4).
The choice of this topic for the doctoral thesis is justified by the importance of
the subject in terms of diabetic foot pathology, given its specific features and the special
impact on the patient's quality of life, as well as on the costs of health services. It is very
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necessary to identify certain factors associated with the risk of complications in the foot
and amputations, as well as to establish parameters that can lead to these results, both for
the subsequent clinical evolution of the patient and to prevent increased cost of care.
These issues are important for all specialists involved, but are especially relevant from the
point of view of a prevention strategy, a way to prevent the occurrence of injuries.
The purpose of this study is to evaluate the factors that influence the severity and
evolution of the complicated diabetic foot, as well as to analyze the survival rates of these
patients and to provide information about predictive factors (positive or negative) for the
evolution of ulcers.
Research objectives
The main objective of the study is to evaluate the factors that may influence the
evolution of diabetic foot ulcers and the prognosis in diabetic patients who have been
hospitalized for various complications of diabetic foot.
The specific objectives in this study are:
- evaluation of the socio-demographic, clinical, biochemical characteristics of
hospitalized patients for complications of the diabetic foot;
- characterization of these ulcerations from an anatomical point of view (location,
topographic aspects, number of affected areas, depth, surface), aggravating factors
(infection), appearance (arteriopathic, neuropathic or mixed), staging using different
classifications of diabetic foot;
- establishing the correlations between the biochemical parameters and the staging of
the diabetic foot;
- establishing the predictive factors for the evolution of diabetic foot complications;
- estimation of survival rates and evaluation of mortality predictors in the studied
group.
4.2. Material and methods
Study group
The present study is an observational, retrospective study, performed on patients
who were hospitalized in the Diabetes, Nutrition and Metabolic Diseases Clinic within the
County Emergency Clinical Hospital "St. Spiridon ”, Iași, between 01.01.2007 -
31.12.2017, having as reasons for hospitalization diagnostics of the diseases in relation to
the pathology of the diabetic foot.
The inclusion criteria for data collection were: patients> 18 years of age,
hospitalized during the above-mentioned period in the Clinic of Diabetes, Nutrition and
Metabolic Diseases, by emergency hospitalization, scheduled or transfer from other
clinics, regardless of type of diabetes (type 1, type 2 or secondary), in which a disease
code corresponding to the diseases included in the pathology of the diabetic foot was
identified in the discharge diagnoses. The first hospitalization for a patient was
considered, if there were several hospitalizations during this period. The formed group
was subsequently tracked, in terms of survival reporting until 2020.
Data collection and management; factors studied
An initial database was created, with the help of the hospital's statistical service,
which contained all patients whose discharge contained codes specific to diseases that
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could be related to the pathology of the diabetic foot (ulceration, gangrene, infections of
the skin and cell tissue sucutaneous, lower limb cellulitis, peripheral arterial disease,
peripheral neuropathy, etc.). From the multitude of patients, patients who did not have a
diagnosis of diabetic foot complication were excluded by the punctual analysis of
discharge diagnoses. Then the hospitalizations for the same CNP were analyzed and the
first hospitalization was selected. Thus, a number of 659 patients were entered in the
database. In order to create the database, the following data were extracted: information
given by the statistical service: age, sex, environment of origin, hospitalization type,
discharge status, discharge type, date of diabetes diagnosis, duration of diabetes, date of
death; information extracted from discharge diagnoses: year of hospitalization,
observation sheet number, type of diabetes, presence of chronic complications;
comorbidities; information extracted from epicrisis: treatment, previous amputations
limited / extended on the affected leg or the other, cellulite, fever, osteolysis (on
radiography), wound secretion, blood cultures; diabetic foot staging: the Wagner-Meggit
classification (5), the University of Texas classification (6), and the San Elian Ulcer
Scoring System (SEWSS) (7). The biological data collected consisted of: complete blood
count, fibrinogen, C-reactive protein, glycemia, glycated hemoglobin, lipid profile: total
cholesterol, triglycerides, LDL-cholesterol, HDL-cholesterol, serum iron, ferritin, urea,
creatinine, glomerular filtration rate by the CKD-EPI method, alkaline reserve, serum
sodium, serum potassium, aspartate aminotransferase, alanine aminotransferase, total
protein, albumin, uric acid.
Statistical analysis of data
Data analysis was performed using SPSS software version 20 (IBM Corp.
Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM
Corp). We analyzed the differences using one-way variance analysis (one way ANOVA),
for homogeneous variables. In the case of inhomogeneous variables, we used a non-
parametric test: the Mann-Whitney U test for 2 groups or the Kruskal-Wallis ANOVA for
more than 2 groups. In the survival analysis, the Hosmer-Lemeshow test, the Kaplan-
Meier curves and the log-rank test were used.
4.3. Results
The study group included 659 subjects, of which 435 men and 224 women. The
mean age (years) ± SD is 61.34 ± 11.06 years, with a minimum of 19 years and a
maximum of 86 years.
Regarding the diagnosis of diabetes, among the patients studied, 12.4% have type
1 diabetes, 86.19% have type 2 diabetes and only 1.67% have other types of diabetes.
Regarding the duration of diabetes, we found an average of 17.59 years for type 1, 10.37
years for type 2 and 10.09 years for other types of diabetes. Metabolic control was poor,
with 77.5% showing an increased glycated hemoglobin value outside the target.
Regarding microangiopathic complications, 65.4% had the diagnosis of retinopathy,
diabetic nephropathy was present in 31.9% of cases. Lower limb obliterative arteriopathy
was diagnosed in 36.4% of patients studied, and peripheral sensory-motor polyneuropathy
in 93.9% of patients in the study (61.3% men and 32.6% women). Analyzing the
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autonomic neuropathy, we identified 11.5% of cases with this complication. Charcot's foot
was identified in 3.2% of cases. Cardiovascular disease was present in 40.2% of cases.
According to the TEXAS classification, we observed that the fewest cases were
in stage AIII (ulcerations that penetrate the bones or joints, without infection, without
ischemia) and B0 (pre and post ulcerative lesions completely epithelialized, with
infection, without ischemia), 0.2% each, and the highest percentage, 21.2% of all patients,
was observed in stage BI (superficial ulcerations that affect the epidermis or epidermis
and dermis, but which do not penetrate the tendons, capsules or bone, with infection
present). According to the Wagner classification, we can see that the highest percentage of
patients 32.4% fall into grade 1 (superficial ulcers), followed by 29.2% in grade 2 (deeper
ulcers that penetrate to the tendons, joints), and the most few cases fall to grade 0 (skin
intact, no lesions).
The average length of hospitalization in the studied group was 19.68 ± 13.38
days. Most patients in the study were hospitalized for <40 days. Subjects who were
classified in stages BII to DIII of the Texas classification had a longer average length of
hospital stay (14.4 days), statistically significant, compared to the groups in the early
stages (9.8 days). Regarding the length of hospitalization in relation to the severity of the
infection classified according to SEWSS and IDSA, we noticed a statistically significant
difference, namely those with severe infection had a longer length of hospitalization. The
length of hospitalization varied statistically significantly in relation to cellulite. Those
with cellulite above the foot lasted longer. Analyzing the length of hospitalization in
relation to the presence of osteolysis, we found that those with osteolysis had a
statistically significantly longer average length of hospitalization than those without (22.6
days vs. 18.3 days). Subjects with the presence of gram-negative aerobes in the wound
had a statistically longer average length of hospitalization compared to the other groups.
The evaluation of SEWSS gradation (mild, moderate and severe), compared by
sex, revealed a percentage of 85.7% moderate degree cases, 9% severe degree cases and
5.3% mild degree cases, without statistically significant differences between sexes (p =
.625) .
There were 278 cases of deaths during the study period (42.2%), as we see in
table 4.65. The average survival time was 9 years, the median being 12 years. Regarding
the general characteristics of the studied group (age and data related to sex, environment,
duration and diabetes, treatment of diabetes), we found statistically significant differences
regarding the survival curve only for the age categories, those over 65 years of age having
a shorter average survival time.
Regarding the complications of diabetes, we found that those with renal
impairment (diabetic nephropathy or chronic end-stage renal disease) had an average
survival time, statistically significantly shorter than the rest of the categories. Moreover,
we found statistical significance in the calculation of survival according to cardiovascular
disease and hypertension, those with these diseases having a shorter average survival time.
The presence of gram-negative aerobes in the wound was associated with a shorter
survival time. In subjects with severe ischemia, we identified a lower average survival
compared to the other stages. Subjects in the Texas classification from BII to DIII had a
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statistically significantly shorter average survival time than those in stages 0 through BI
(8.5 years vs. 10 years). Grades 3,4,5 in the Wagner classification were associated with a
lower average survival than the first 2 grades (8.4 vs. 9.2 years). Charcot osteoarthropathy
has been associated with a statistically significantly shorter average survival time.
Variables that predict survival were analyzed by univariate Cox regression. In
this analysis, the main factors that contributed to the increase in mortality were: length of
hospitalization, diabetic nephropathy, chronic kidney disease, glomerular filtration rate
below 60ml / min / 1.73m2, cardiovascular disease, hypertension, low hemoglobin (<10g
/Mister). In the multivariate analysis, the adjustment was made with potential confounding
factors that could compete on the prognosis of patients according to the results obtained
from the univariate analysis: age, sex, duration of diabetes, association with
cardiovascular disease, glomerular filtration rate (<60 ml / min / 1.73 m2 ), anemia (Hb
<10 g / dl), serum albumin.
To achieve this goal, the Texas classification was divided into 2 categories (from
stage A0 to BII one category and from BII to DIII second category. Patients with deep
ulcers with damage to tendons or osteoarticular elements presented a higher risk of high
death rate compared to those who had superficial or preulcerative lesions at the first
presentation (HR = 1,963, 95% CI: 1,065-3,617) and after taking into account the factors
that could simultaneously influence the duration of survival. predictive effect of
associated cofounders (age, cardiovascular disease HR = 2.89, anemia HR = 1.28,
glomerular filtration rate HR = 2.17).
Patients with bone or gangrene damage at hospitalization had a 88.9% higher risk
of death during follow-up (CI 1,024 - 3,483) compared with those with ulcers without
bone or joint damage after inclusion in the disease prediction model. cardiovascular
disease, anemia, impaired renal function and demographic factors. Among the main
factors taken into account in the SEWSS assessment, those that contributed statistically
significantly to the increase in mortality were: lesion topography (lateral or medial),
ischemia and SEWSS score.
4.4. Discussions
Diabetic foot ulcer is one of the common complications of diabetes and can be an
independent risk factor for amputation, accompanied by high health costs and sometimes
even death. The group studied by us included a number of 659 subjects, patients with
diabetes and diabetic foot, a group in which the male gender predominated (66% men),
the environment of origin being in most cases urban, and the average age of patients was
61.34 ± 11.06 years, female patients being older compared to male patients (p <0.001).
The results obtained in our study in terms of gender distribution are consistent
with other studies in which males predominated. Thus, in the research conducted by
Ramanathan et al., Of the 100 subjects with diabetic foot ulcers included in the study,
70% were men (8). Also in terms of age, in the study conducted by Ramanathan, the
participants had an average age of 59.91 ± 10.6 years, the majority (78%) being in the age
group between 40 and 70 years. (8).
In terms of type of diabetes, most patients included in our study were diagnosed
with type 2 diabetes (86.19%), and the average duration of the disease was 11.24 years.
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77.5% of patients had poor long-term metabolic control. In another study, the mean
duration of diabetes was 9.66 years (8). Other data in the literature confirm that the
majority of patients with ulcers are patients with type 2 diabetes, the majority of men (9).
The data obtained in our group show that, if we take into account the criteria of
the Wagner classification, the highest percentage of patients (32.4%) falls to grade 1
(superficial ulcers), followed by 29.2% in grade 2 (deeper ulcers that penetrate up to the
level of tendons, joints), and the fewest cases fall to grade 0 (intact skin, no lesions). This
classification has many disadvantages such as the fact that only one of the 6 degrees
includes infection. At the same time, this system is limited in terms of identifying and
describing vascular damage (10) and is based mainly on criteria of ulcer depth and tissue
viability, not taking into account neuropathic damage, with loss of protective sensitivity.
Also, this system cannot differentiate an ischemic ulceration from an infected one (11).
Subjects in whom gram-negative aerobic bacteria were identified in the wound had a
longer average duration of hospitalization, statistically significant, compared to the other
groups (p <0.001). Coagulase-negative staphylococcus isolated from diabetic foot ulcers
has been correlated with ulcer severity (12).
Patients with renal impairment (diabetic nephropathy or chronic end-stage renal
disease), cardiovascular disease, or hypertension had a shorter survival time. The presence
of gram-negative aerobes in the wound was associated with a shorter survival time.
Subjects classified in the Texas classification in stage B - grades II, III, stage D - grades 0-
III had a shorter average survival time than those in stages A0-III and BI (8.5 years vs. 10
years). Grades 3,4,5 in the Wagner classification were associated with a lower average
survival than the first 2 grades (8.4 vs. 9.2 years). Patients with bone or gangrene damage
at the time of hospitalization had a higher risk of death during follow-up, by 88.9% higher
compared to those with ulcers without bone or joint damage. The main factors that
contributed to the increase in mortality were: length of hospitalization, diabetic
nephropathy, chronic kidney disease, glomerular filtration rate below 60ml / min /
1.73m2, cardiovascular disease, hypertension, low Hb (<10g / dl).
Regarding the long-term survival of patients with diabetic foot, there are
numerous data in the literature. Thus, Aragon-Sanchez et al. analyzed a retrospective
cohort of 150 patients with diabetic foot infections who underwent surgical treatment in
order to evaluate long-term outcomes. The group was followed for a median period of 7.6
years (13). In the group studied by us, patients with deep ulcers with damage to tendons or
osteoarticular elements had a higher risk of death compared to those who at the first
presentation had superficial or pre-ulcerative lesions (HR = 1,963, 95% CI: 1,065 -3,617)
and after taking into account the factors that could simultaneously influence the duration
of survival.
4.5. Conclusions
In our group, most patients had type 2 diabetes (86.19%), with a mean disease duration
of 11.24 years. Most patients (78.1%) had a poor glycemic control (objectified by
HbA1c> 7%), with a mean HbA1c value of 9.5%.
Most patients had peripheral diabetic sensory-motor polyneuropathy (93.9%), and
about a third had peripheral arterial disease. According to the Texas classification, the
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highest percentage of patients were in stage B grade I (19.3%). According to the
Wagner classification, the highest percentage of patients were in grade 1 (32.4%).
The average length of hospitalization in the studied group was 19.68 ± 13.38 days and
was longer in those with a history of amputations, in those who were classified in
stages BII to DIII of the Texas classification, in those with severe infection, with
cellulite present, with osteolysis and in subjects in whom the culture in the wound
showed gram-negative aerobic bacteria. The length of hospitalization was positively
correlated with the parameters of inflammatory status and negatively with the value of
hemoglobin, hematocrit, total protein and serum iron.
We found that patients with osteolysis had higher mean blood glucose, HbA1c,
leukocytes, neutrophils, platelets, fibrinogen and ferritin, and lower mean hemoglobin
and hematocrit than those without osteolysis.
When analyzing the degrees of SEWSS score in relation to other parameters, we found
that patients with severe ulceration had lower mean hemoglobin, hematocrit and
sideremia and higher mean values for most markers of inflammation (leukocytes,
neutrophils, platelets, fibrin , C-reactive protein).
There were 278 deaths during the study period (42.2%), with a mean survival of 9
years.
Patients with renal impairment (diabetic nephropathy or chronic end-stage renal
disease), cardiovascular disease, or hypertension had a shorter survival time. The
presence of gram-negative aerobes in the wound was associated with a shorter survival
time.
Subjects in the Texas classification from BII to DIII had a shorter average survival
time than those in stages 0 through BI (8.5 years vs. 10 years). Grades 3, 4, 5 of the
Wagner classification were associated with a lower average survival than the first 2
degrees (8.4 vs. 9.2 years).
Patients with bone or gangrene damage at the time of admission had a 88.9% higher
risk of death during follow-up compared to those with ulcers without bone or joint
damage
Among the main factors taken into account in the SEWSS assessment, those that
contributed statistically significantly to the increase in mortality were: lesion
topography (lateral or medial), ischemia and severity of ulceration, assessed by the
final score of the SEWSS. The main factors that contributed to the increase in
mortality were: length of hospitalization, diabetic nephropathy, chronic kidney
disease, glomerular filtration rate below 60 ml / min / 1.73m2, cardiovascular disease,
hypertension, low hemoglobin (<10g / dl) .
Chapter 5. Observational analytical study to assess bone turnover in patients
with type 2 diabetes using modern biomarkers
5.1. Motivation and objectives of the doctoral study
Diabetic foot ulcers are the most common complication of patients with diabetes.
However, studies show that when there are already clinically obvious chronic
complications, the disease is already advanced, and so the therapeutic possibilities are
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often limited. It is therefore useful to have the possibility of early identification of patients
with diabetes at risk of developing complications in the foot, in order to provide a real
chance to prevent disabling ulcers. The bone structure at the level of the foot is perhaps
the least taken into account when it comes to classic risk scores. However, the quality of
the bone structure in the foot is relevant both in what it can suggest about the
pathophysiology of foot complications and in what it can show about the patient's
evolution and prognosis. Bone turnover is a balanced relationship between the process of
bone formation by osteoblasts (creation of new bone) and the process of bone resorption
by osteoclasts (removal of old bone), in which all sequences are fine-tuned by a multitude
of markers. of the two processes. Two markers of bone turnover seem promising in the
roles they play in this context: RANKL and FGF23. Identifying the relationship between
these biomarkers and other known parameters (clinical, paraclinical, nutritional or
lifestyle) can help create an algorithm to stratify the risk of progression to ulcers in order
to sort patients who need more prompt interventions. Research objectives The main
objective of this study was to evaluate two modern biomarkers of bone turnover in
patients with type 2 diabetes. The secondary objectives of the present study were to:
characterize the relationships between biomarkers used (RANKL and FGF23) and
clinical, biological, nutritional and lifestyle parameters in patients with type 2 diabetes, as
well as to investigate the predictive value of RANKL and FGF23 in identifying patients
with type 2 diabetes who are at risk of developing bone complications.
5.2. Material and methods
5.2.1. Study group
The present study is an observational, analytical study performed on patients with
type 2 diabetes who presented for the annual evaluation of complications and
comorbidities by day hospitalization, at the Clinical Center for Diabetes, Nutrition and
Metabolic Diseases within the Emergency Clinical Hospital "St. Spiridon" Iasi during a
year (2017) and who signed an informed consent. The inclusion criteria were patients with
type 2 diabetes (with or without insulin treatment), no history of chronic kidney disease,
Charcot's arthropathy or other forms of autonomic neuropathy or a history of amputation
due to diabetic foot. We excluded patients with psychiatric or neurological disorders
associated with cognitive impairment, active forms of cancer, diseases associated with
immobilization, and patients who refused to participate.
5.2.2. Data collection and management; factors studied
We performed a comprehensive evaluation of the patients included in the study,
which included general data, measurement of anthropometric parameters, clinical
evaluation by history and clinical examination (but also by analyzing existing data in the
dispensary of these patients), paraclinical evaluation and style evaluation life through
validated questionnaires. The following biomarkers were evaluated as part of the annual
patient check: fasting blood glucose, glycated hemoglobin, urea and creatinine (eGFR was
calculated using the CKD-EPI formula), ALT and ASAT, total cholesterol, HDL-
cholesterol, LDL-cholesterol, triglycerides, uric acid, hemoglobin and hematocrit, white
blood cells and platelets. As part of the nutritional assessment, we assessed serum levels
of: total protein, albumin, calcium, magnesium, iron, vitamin D and vitamin B12. We
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evaluated the serum level of RANKL and FGF23 using a third generation enzyme
immunoassay technique for RANKL and a multi-matrix ELISA technique for FGF23 (C-
Terminal). We evaluated patients 'regular food and nutrient intake in the last 12 months,
using the EPIC food frequency questionnaire, previously validated in Romania (14) and
introduced patients' responses to the EPIC food frequency questionnaire in FETA, a
software created for this use ( 15). We evaluated the usual physical activity of patients
using the International Physical Activity Questionnaire (IPAQ) long form, translated into
Romanian according to the rules for translating validated questionnaires into another
language (16). Statistical analysis of data I used SPSS Statistics version 20 for data
analysis. We analyzed the differences in averages between two or more continuous
variables using unidirectional ANOVA (if the variables were found to be homogeneous;
otherwise, we used a non-parametric test: the Mann-Whitney U test for 2 samples or
Kruskal -Wallis 1- way ANOVA for more than 2 samples). We used Pearson linear
regression correlations (or Spearman correlations for ordinal variables) to analyze the
associations between the variables.
5.3. Results
The group included 171 patients, of which 83 (48.5%) were men. The mean age
of the patients in the study group was 60.88 ± 10,125 years. Regarding the area of origin,
the frequency of cases in urban areas was 70.2% (120 cases) and 29.8% (51 cases) in rural
areas, without statistically significant differences. In terms of occupation, most patients
were retired (42.7%). The patients included in the study had a mean duration of diabetes
of 7.7 years. The presence of diabetic retinopathy was found in 11.1% of the study group.
Peripheral diabetic neuropathy was present in 36.8% of subjects in the analyzed group.
We identified a 2.4% percentage of subjects who had obliterating arteriopathy of the
lower limbs. Macroangiopathy was present in 14.6% of the patients evaluated in the study.
We analyzed the values of nutritional markers for the whole group and separately by sex.
We found that the mean value of albumin and vitamin D in men was statistically
significantly higher than in women. The calculation of the correlations between the food
groups and the parameters of bone turnover highlighted the following: RANKL values
were positively associated (strong correlation) with the consumption of fish, nuts and
seeds and negatively associated (strong correlation) with the consumption of potatoes;
statistically weaker associations between RANKL and the consumption of non-alcoholic
beverages (direct combination) and the consumption of soups and sauces (reverse
combination); a weak inverse association between FGF23 and fish consumption. We have
shown that FGF23 is negatively associated with the duration of diabetes. When comparing
the mean values of RANKL and FGF23 between the groups treated with metformin vs.
without metformin, we did not find statistically significant differences. We did not find
significant differences when comparing bone parameters by groups of categories
according to incretin treatment. The averages of RANKL and FGF23 levels were
compared between all categories of subjects according to the treatment received, but no
statistically significant differences were found. We comparatively analyzed the mean
RANKL and FGF23 in patients grouped according to the presence / absence of
retinopathy, highlighting that the average serum levels of FGF23 is lower in the group
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with retinopathy present, although these data are not very statistically significant.
Analyzing the data further, we found that patients with neuropathy have lower serum
levels of RANKL and FGF23, compared to those without neuropathy. We did not find
statistically significant differences when comparing bone parameters, by groups
depending on the presence / absence of arteriopathy. The group of patients with
macroangiopathy had a statistically significantly lower RANKL average than patients
without macroangiopathy. Analyzing the correlations between glycemic control
parameters and those of bone turnover, we highlighted a strong inverse association
between FGF23 with glycemia and HbA1c, which indicates that the higher the latter, the
lower the FGF23 values. The group of patients with good glycemic control (HbA1c <7%)
had a statistically significantly higher mean FGF23 values compared to the group with
poor glycemic control (HbA1c> 7%). RANKL averages did not vary significantly
between these groups. The calculation of the correlations between the lipid profile and the
bone parameters revealed a strong inverse association between triglycerides and FGF23,
which indicates that as the triglyceride value increases, FGF23 decreases. We also found
inverse associations between FGF23 with LDL cholesterol and uric acid, but weak,
without statistical significance. In the analysis of the correlations between renal and
hepatic parameters with bone turnover parameters, we found the following: positive
associations between FGF23 with creatinine and direct bilirubin; inverse association
between FGF23 and glomerular filtration rate; inverse associations between RANKL and
TGP, respectively TGO. These data indicate that FGF increases as creatinine increases
and eGFR decreases, concluding that FGF23 is higher in kidney disease. Also, the lower
the transaminase levels, the higher the RANKL. Further analyzing the correlations
between inflammatory and hematological parameters with bone turnover parameters, we
highlighted a statistically significant inverse association between FGF23 and Hb and a
statistically significant direct association between FGF23 and VEM. These data suggest
that the higher the FGF23, the lower the Hb and the higher the VEM. We notice a
statistically significant inverse correlation between total proteins and serum albumin with
FGF23 levels.
5.4. Discussions
In our group of patients with type 2 diabetes we did not find a correlation
between the RANKL level and the duration of the disease, the level of glycemic control
(evaluated by blood glucose value and HbA1c), the parameters of lipid or protein
metabolism. RANKL was lower in patients with type 2 diabetes and chronic
complications such as peripheral neuropathy and macroangiopathy, and in those with
abdominal obesity. Regarding the association of RANKL level with various aspects of
lifestyle, the results obtained by us in the study group show that RANKL level was lower
in patients who reported higher alcohol consumption, and was higher in those who
reported that they walk more, as well as those who had a higher intake of fish, nuts and
seeds. The involvement of the OPG / RANK / RANKL system in the pathogenesis of DM
has been investigated and appears to have a potential role as blocking this system has
improved hepatic insulin resistance and prevented the development of DM (17). Studies in
mice with type 2 diabetes show that systemic or hepatic blockade of RANKL signaling
13
has led to a marked improvement in hepatic insulin sensitivity and improved blood
glucose (18). The decrease in RANKL level observed in the literature has been attributed
to immature osteoblasts and an increase in the number of osteoclasts (19). Hyperglycemia
is cytotoxic to osteoblasts by inducing apoptosis, resulting in reduced bone formation
(20). The results of a study by Panezai et al. indicates that hyperglycaemia significantly
alters OPG and RANKL levels, thereby disrupting bone resorption mechanisms in type 2
diabetes. The authors found that serum RANKL levels were lower and OPGs higher in
patients with diabetes compared with nondiabetics and prediabetics. Our results show that
the level of FGF23 decreases with increasing DZ duration, being inversely proportional to
the value of blood glucose and HbA1c. However, the results are controversial in the
literature. Thus, in some studies, DM was correlated with higher circulating FGF23 levels
(22, 23), while other studies did not find these correlations (24). The results of our study
showed the existence of a relationship between RANKL level and the presence of
peripheral neuropathy and macroangiopathy, in these patients the RANKL level being
lower. In a recently published study, it was shown that in patients with diabetic foot the
concentration of FGF23 was significantly higher compared to patients diagnosed with
diabetes, but without diabetic foot (23.8 (17-32.2) vs 15.5 (10.1-24.5) pg / mL , p <0.01).
Compared to the level obtained in the group of patients without diabetic foot, in those
with diabetic foot there is an increase of FGF23 by 30.3% (25). It should be mentioned
that in the studied group there were a small number of patients with peripheral
neuropathy, early stages of the disease. Patients with advanced stages of the disease or
Charcot's neuroarthropathy were not included in the study group, most of the data in the
literature being reported especially for these categories of patients. In conclusion, we
conducted an analytical study in which we included, consecutively, all patients who met
the inclusion and exclusion criteria mentioned. For this reason, the study population was
quite heterogeneous in terms of disease duration, level of glycemic control, the presence
of chronic complications or the type of antidiabetic treatment used. The aim of the study
was to evaluate the predictive value of RANKL and FGF23 in the early stages of diabetic
foot lesions, which is why patients with Charcot neuroarthropathy or a history of
amputations were excluded. This could explain the differences between the results
obtained in the studied group and the data published in the literature. These markers of
bone turnover usually change in situations characterized by intense processes of bone
turnover, such as the diabetic foot or even the Charcot's foot, especially in its acute phase
when the processes of catabolism and inflammation are intense. In fact, studies following
and monitoring these types of patients assess the dynamics of changes in plasma levels of
these parameters compared to the acute phase. Usually, as we have shown, the RANKL
level is increased in the acute phase of the diabetic foot so that later, in evolution, it
decreases until stabilization. In the studied group, the level of glycemic control was
satisfactory and the duration of the disease was relatively short compared to what is
usually reported in the literature in relation to the level of RANKL and FGF23. In the
study group, the mean value of HbA1c is 7.1 ± 1.3%, compared to an average of 8.01 ±
1.9% mentioned in the literature, and with a mean duration of diabetes of 7.7 ± 6.7 years
in our group compared to 13.3 ± 7.6 years mentioned in most studies (26).
14
5.5. Conclusions
In the study group, the mean age of the patients was 60.88 years, with a mean duration
of type 2 diabetes of 7.7 years and a mean HbA1c value of 7.1%.
Most patients had a degree of overweight at inclusion in the study, about a third of
patients with peripheral sensory-motor polyneuropathy.
The average daily intake of macronutrients in the total batch was 205.9 grams of
carbohydrates (of which 18.48 grams of fiber), 82.24 grams of protein, 60 grams of fat
(of which 21.3 grams of monounsaturated, 21.5 grams of polyunsaturated and 20.69
grams of saturated). Women had a higher average intake of total lipids and
polyunsaturated lipids than men.
In patients with neuropathy, there were fewer minutes sitting and fewer hours of
sleep/night compared to those without neuropathy. The value of blood glucose was
inversely correlated with the intake of calories, protein, monounsaturated and saturated
lipids, vitamin D, intake of nuts and seeds and sweets.
We found a direct correlation between the duration of diabetes and glycemic control
parameters. We did not find significant differences when comparing markers of bone
turnover between the sexes.
Age was positively associated with FGF23. We found that FGF23 was negatively
associated with the duration of diabetes. Serum FGF23 levels were higher in patients
with anemia and those with good glycemic control. We found an inverse association
between FGF23 and blood glucose, HbA1c, hemoglobin, total protein and serum
albumin, as well as a direct association between FGF23 and mean erythrocyte volume.
We found that there was an inverse correlation between the abdominal-buttock index
and the RANKL value. Patients with macroangiopathy had lower RANKL levels. The
mean RANKL and FGF23 values were lower in smokers and those with neuropathy.
We did not find associations between the average daily intake of minerals and
vitamins and the values of RANKL and FGF23.
Chapter 6. General conclusions
The thesis consists of a retrospective and a prospective study on osteo-articular
changes in diabetic patients.
In the retrospective study, which included patients with diabetic foot ulcers:
o The average survival time was 9 years, being shorter in those with renal
impairment (advanced and chronic chronic renal nephropathy and advanced renal
disease), cardiovascular disease, hypertension, wound infection (gram-negative
aerobic bacteria), with higher severity of ulceration, according to W system;
o Patients with bone or gangrene damage at the time of admission had a higher
risk of death during follow-up, 88.9% higher compared to those with ulcers without
bone or joint damage;
o The main factors that contributed to the increase in mortality were: lesion
topography, ischemia and severity of ulceration (assessed by the final score of
SEWSS), length of hospitalization, diabetic nephropathy / chronic kidney disease with
glomerular filtration rate below 60 ml/min/1,73m2, cardiovascular disease,
15
hypertension, anemia (hemoglobin <10g / dl).
In the prospective study, which looked at early biomarkers of bone damage in diabetic
patients:
o The mean values of RANKL and FGF23 were lower in subjects at risk of
developing diabetic foot, ie in smokers and those with neuropathy, macroangiopathy
and higher mean values of FGF23 were found in patients with anemia.
o It was found that there is an inverse correlation between abdominal-buttock
index and RANKL value and between FGF23 and blood glucose, HbA1c, hemoglobin,
total protein and serum albumin, as well as a direct association between FGF23 and
mean erythrocyte volume.
Both studies provide arguments for the need for regular assessment of risk factors for
diabetic foot that could prevent complications and increase survival.
Chapter 7. Aspects of originality and perspectives that the thesis opens
To our knowledge, the retrospective study is the first study in the region of
Moldova to analyze a period of 10 years of hospitalization of the diabetic foot
complicated and thus brings important data in the epidemiology of the diabetic foot in
Romania. Also an element of originality is the analysis of survival in their patients. The
observational analytical study is also a first, as it evaluates some modern biomarkers of
bone turnover, in patients with type 2 diabetes, with good glycemic control, with few
chronic complications, in the idea of trying to identify those patients at risk. for diabetic
foot changes. The dosage of these markers in this group of patients is of major importance
for further research, in order to create a cohort and monitor the evolution over time of
these parameters. From the retrospective observational study, the following directions for
further research are opened: extension of the study group: to include hospitalizations from
surgery clinics or extension of the research period; conducting a prospective study in
which it will be possible to evaluate parameters of interest that were not accessible in this
study (evaluation of nutritional status, evaluation of other lifestyle parameters, description
of socio-economic parameters, detailed analysis of aspects related to gait biomechanics
and data related to specific therapeutic education for foot care). From the observational
analytical study the following directions of further research are opened: the extension of
the study group (increasing the number of subjects would increase the statistical power of
the results, and the extension of the group could be done in patients with type 1 diabetes,
to evaluate the particular evolution of these patients); evaluation of other parameters, in
particular other parameters of bone turnover or phospho-calcium balance, in order to be
able to describe more precisely the role of RANKL and FGF23 in the evolution of bone
complications in these patients; tracking this group over time would allow obtaining real
data on the predictive value of modern biomarkers used, thus being able to achieve an
algorithm for early stratification of the risk of ulcers in patients with diabetes.
16
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18
Niță George - List of published works from the doctoral thesis
Articles in extenso in BDI indexed journals
Niță G, Gherasim A, Niță O, Popa AD, Burlui AM, Arhire LI, Mihalache L, Graur M.
Diabetic foot and periodontal disease: possible common pathogenic ways related to bone
turnover.Int J Med Dent 2021; 25 (2): 43-52.
Niță G, Gherasim A, Niță O, Popa AD, Arhire LI, Mihalache L, Graur M. Analysis of
factors influencing the duration of hospitalization in patients with diabetic foot
ulcers.Rom J Med Pract 2021; 16 (2).DOI: 10.37897 / RJMP.2021.2.21.
Articles in extenso in ISI indexed journals
Niță G, Niță O, Gherasim A, Arhire LI, Herghelegiu AM, Mihalache L, Tuchiluș C,
Graur M. The role of RANKL and FGF23 in assessing bone turnover in type 2 diabetic
patients.Acta Endocrinol (Buc) 2021;17 (1).doi: 10.4183 / aeb.2021.X
- article accepted for publication