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