The Role of Mitochondria Dynamic in bone healing
Abstract
The regeneration capacity of tissues can best be monitored after injury; therefore, bone
healing was in the focus of several studies as a model system of regeneration. Bone is a
living tissue that not only mechanically supports the body and protects vital organs, but also
produces blood cells and stores minerals. As a result of aging, hormonal imbalances,
nutrient deficiencies, or the frequent use of certain medications, the bone remodeling cycle
process may become unbalanced. Like many other European societies, the German
population is an aging society. Recently the population development department of the
Federal Statistics Office predict that by the year 2040, one in two of German population will
be older than 50 and one in three older than 60 years old. One in every seven will be over
the age of 80 years old.In aged patients, the capacity of such regeneration is limited. Many
studies have addressed several factors that correlate with aging along and with the energy
metabolism, however, rarely were mitochondria the target of investigations. Mitochondria
are powerhouses of the cell, generating chemical energy in the form of ATP. Several agerelated pathologies like neurodegenerative or metabolic diseases and cancer have been
linked to mitochondria. Today we know that the initial phases of healing turn from proinflammatory state into anti-inflammatory state. Our knowledge also revealed that
inflammation induce mitochondrial dysfunction. Recent studies even showed promising
results by utilizing fission inhibitors to encounter mitochondrial dysfunction.
Simply put, balancing the dynamic of mitochondrial morphology is possible and have
potential in understanding the limited regenerative capacity at aging. However, little is
known in this specific regard. The project will deepen our knowledge of mitochondrial
dynamic differences between cells from old and young donors, and can offer answers to
the continuous downregulation of mitochondrial genes throughout the healing process. The
project will also unravel the effects of pro- and anti-inflammatory stimulants on stromal cells
and their differentiation. Furthermore, the crosstalk between mitochondrial dynamics and
strontium as a prophylactic drug will be investigated. We hypothesized that treatment with
non-steroidal anti-inflammatory drugs (NSAID) and Strontium will both contribute to the
balance in mitochondrial dynamics. Furthermore, we also hypothesized that fission
inhibitors will reflect favorably on osteogenic and chondrogenic differentiation of stromal
cells from aged donors.
Keywords
Bone, Mitochondria (Mt), Mitochondrion Dynamics, Mesenchymal Cells (MSC),
Osteogenesis, Age related diseases
Introduction
One important neglected factor is the weakness of tissue regeneration in later ages. The
ability of the bone to restore its structure and function after injury declines, even in healthy
old individuals. To best understand the process of bone healing firstly we must be able to
understand the molecular mechanisms by which the bone is generated from its precursors
stem cells. This can be explained by focusing in the mesenchymal stem cells (MSCs)
differentiation towards bone tissue [1, 2]. Furthermore, this will enhance not only our
understanding of aging but also most importantly the possible therapy for old patient.
Bone fractures like and other body injury, once its occur, it will directly stimulate the immune
system, since the bone is highly vascularized tissue. At the early stages of healing, bone
hematoma moves from the pro-inflammatory into an anti-inflammatory state[9].
Furthermore, low levels of oxygen and glucose as well as high concentrations of lactate and
reductive metabolites. We know that the inflammation caused by the immune response and
the bioenergetics situation together have a considerable relationship with the mitochondrial
activity in bone healing process. Mitochondria does not only produce energy as ATP units,
but also play a critical role in the regulation of inflammation, metabolism and energy supply
[11]. One more feature about the mitochondria is called the mitochondrial dynamic which
characterized by the ability to fission (Divide) and or fuse (Merge) as a quality control
process to maintain the intracellular homeostasis environment surrounding the nucleus.
Aging disrupts fusion and fission cycles leading to changing the normal behavior inside the
cell [13].
Taken together, understanding the correlation of mitochondria dynamics to the changing
pro- and anti-inflammatory state of hematoma might be a key to understand the differences
between cell behaviors in aged and young patients. New findings by our collaborator PD.
Dr. med, Matthias Hecker of the department of internal medicine II at the university hospital
Giessen showed that 18R-HEPE and Resolvin E1 (RvE1) possess anti-inflammatory and
anti-apoptotic properties. They can restore inflammation-induced mitochondrial dysfunction
by decrease in mitochondrial respiration and membrane potential, furthermore, imbalance
of mitochondrial fission and fusion [14]. Fission is controlled by dynamin related protein 1
(Drp1) [15]. Furthermore, inhibition of mitochondrial fission by Mdivi-1 and Dynasore
reduces levels of the pro-inflammatory cytokines IL-6 and IL-8. That means we can control
the mitochondrial dynamic (fission/fusion) in vitro proportional to inflammation. Furthermore,
this is indicative of the potential that lies in investigating the mitochondrial dynamic in aging.
In this project, we will collaborate closely with PD. Dr. Hecker as an expert in the field.
Furthermore, osteoporosis is one of the most common age-related disease, were fractures
are considered a major cause of increased mortality. Therefore, aging patients are
encouraged to take prophylactic drugs capable of promoting bone formation and,
simultaneously inhibiting bone resorption. Long-term studies showed that Strontium
ranelate is an effective compound for chronic treatment of osteoporosis-mediated bone loss
[16]. Several studies also showed the efficacy of locally applied strontium on healing of
osteoporotic fractures [17-24]. Intriguingly, reports about strontium interaction with
mitochondria without exerting a damaging effect as seen with calcium ions [25].
MMMMMMMMMMMMMMMMMMMM
Therefore, we will examine the mitochondrial dynamics in mesenchymal stromal cells from
aged and young donors under following hypotheses:
1- Young mesenchymal stromal cells show enhanced fission dynamics than old cells.
2- Pro-inflammatory treatment (with tumor necrosis factor (TNF-α)) will induce
mitochondrial fission and reduced fusion, which is not favorable for cellular function.
3- Anti-inflammatory treatment (with nonsteroidal anti-inflammatory drugs, Diclofenac
sodium), will induce mitochondrial fusion, which is favorable for the cell function.
4- Stimulating aged cells using Strontium ranelate, will rejuvenate cells from old
patients by shifting mitochondrial dynamics towards fusion and promote genes
regulating energy metabolism.
5- Fission inhibitors (Mdivi-1 and Dynasore) will reverse the effects of pro-inflammatory
treatment and reflect upregulation osteogenesis genes.
Preparatory work done:
The success of fracture healing relies on coordinated overlapping cellular and molecular
events. Bone healing is considered as a model system to understand tissue regeneration
[25]. Thereby, we first retreated to our microarray data to pinpoint mitochondrial irregular
gene expression, whether chronological, temporal or functional changes arises in the
course of pathological bone healing. Therefore, we analyzed the healing progression of
standard closed femoral fracture in C57BL/6N (age = 8 weeks) wild type male mice model.
Fracture callus was assessed at days (D) 3, 7, 10, 14, 21, and 28 post fracture by microarray
and histological analysis. Interestingly, microarray analysis of the essential role of
mitochondrial genes in the success of bone healing showed a significant downregulation of
mitochondrial genes most prominent during the early inflammatory phase. Out of 39
differentially expressed mitochondrial genes, only one single gene was significantly
upregulated at onetime point. Crucial genes like Gpx1, Peroxidase 2 (Prdx2) and
Peroxidase 3 (Prdx3), that play role against oxidative stress condition were downregulated
at D3 and D7. 3-Oxoacid CoA transferase 1 (Oxct1), which plays a central role in ketone
body catabolism was significantly downregulated at D3 and D7. Genes like Solute carrier
family 25 member 11 (Slc25a11), Solute Carrier Family 25 member 20 (Slc25a20), and
Branched chain ketoacid dehydrogenase kinase (Bckdk) that play role in mineral transport
and exchange were significantly downregulated at D3 and D7. Other important genes for
mitochondrial transport and electron transport chain like Transmembrane proteome 14C
(Tmem14c), Atp5b, ATP synthase H+ transporting mitochondrial f1complex (Atp5f1) and
Translocase of outer mitochondrial membrane 7 (Tomm7) were significantly downregulated
at D3 and D7. Genes like Citrate Synthase (Cs), Fumarate Hydratase 1 (Fh1), Succinyl CoA
ligase (Suclg1), Ubiquinol-Cytochrome C Reductase Core Protein 2 (Uqcrc2), and
Cytochrome c1 (Cyc1) that are important for energy producing signaling pathways were
significantly downregulated at D3 and D7.
Coenzyme Q9 (Coq9) which involves lipid biosynthesis for electron transport chain was
significantly downregulated during the early phase. Another downregulated gene was
Cytochrome P450 family 27 subfamily A member 1 (Cyp27a1) which plays a role in keeping
cholesterol homeostasis. Cyp27a1 was significantly downregulated at D3 and D7. Further,
there were erythropoiesis regulatory gene like Heat shock protein family a member 9
(Hspa9) and metal binding gene like Iron-Sulfur Cluster Assembly 1 (Isca1). Hspa9 and
Isca1 were significantly downregulated at D3 and D7. Cold Shock domain containing protein
E1 (Csde1) which acts as RNA binding protein during transcription and translation was
significantly downregulated at D3 and D7.
Further, glucose and fatty acid metabolism is important for bone homeostasis regulated by
gene like Pyruvate Dehydrogenase Kinase 4 (Pdk4), which was significantly downregulated
at D3 and D7. Another important gene is Electron transfer flavoprotein alpha subunit (Etfa)
which is needed for amino acid metabolism. Etfa was significantly downregulated at D3 and
D7. Further, Glutathione S transferase P1 (Gstp1) which links stress kinase and cell
apoptotic pathway was significantly downregulated at D3 and D7. Another downregulated
gene was Translocase of inner mitochondrial membrane 8 homolog B (Timm8b) which acts
as chaperone for protein transport. Timm8b was significantly downregulated at D3 and D7.
Genes like Vdac1 and Voltage Dependent Anion Channel 3 (Vdac3) that perform role in
diffusion and binding of molecules were also downregulated. Vdac1 was significantly
downregulated at D7 and Vdac3 was significantly downregulated at D3, D7, and D21.
Further, Carnitine O-palmitoyltransferase 1 (Cpt1b) which acts as a unit for fatty acid beta
oxidation was significantly downregulated at D10 and D14. Another downregulated genes
were Cytochrome c oxidase subunit 8B (Cox8b), Cytochrome C oxidase subunit 7A1
(Cox7a1), and Cytochrome C oxidase subunit 6A2 (Cox6a2) that are involved in electron
transport activity. Cox8b and Cox7a1 were significantly downregulated at D10 and D14.
Cox6a2 was significantly downregulated at D10. Another important gene was Creatine
kinase S-type (Cktm2) which serves as energy transducer was significantly downregulated
from D3 through D7, D10 until D14. Further, carbohydrate metabolism gene like Succinate
dehydrogenase cytochrome b560 subunit (Sdhc) was significantly downregulated at D14.
Lyr motif containing protein 5 (Lyrm5) which acts as electron transfer flavoprotein regulator
was significantly downregulated at D14. Another important gene was Dual Specificity
Phosphatase 26 (Dusp26) which acts as an inhibitor of Mapk1 and Mapk3. Dusp26 was
significantly downregulated at D14. Heme biosynthesis gene like Solute carrier family 25
member 37 (Slc25a37) was significantly downregulated at D21 and D28. Among
upregulated genes, there was molecule exchange factor like Solute carrier family 25
member 5 (Slc25a5), which was significantly upregulated at D10.
The drastic downregulation of mitochondrial genes during early inflammatory phase urged
the examination of mitochondrial activity at cellular level. Therefore, an antioxidant protein
(GPX1) was localized using IHC (Figure 1). The positive signal of GPX1 was examined
throughout the fractured callus and specifically in the osteocyte vicinity. Descriptively, GPX1
positive signal was seen in small patches within bone marrow and in callus region (Figure
1A). Histomorphometry showed lower GPX1 positive area at D7 when compared with other
time points (Figure 1E). D28 showed higher GPX1 stained area when compared with other
time points (Figure 1E). But no significant differences were noted. On the other side, Silver
nitrate counter-stained sections showed GPX1 signal within osteocytes and around blood
vessels (Figure 1C). GPX1 signal was seen around blood vessels rather than osteocytes at
D7. Further, GPX1 positive signal was seen around blood vessels and some patches within
osteocytes vicinity at D10. Interestingly, D14 showed higher number of GPX1 signal within
osteocytes vicinity than blood vessels (Figure 1C). GPX1 signal was seen in osteocytes
vicinity and around blood vessels at D21. GPX1 signal was not seen within osteocytes at
D28.
Figure 1: Histological analysis of GPX1 and UBB activity during fracture healing showed
active positive signal within bone matrix and bone marrow. (A, A´) D28 showed GPX1 stained
region around the newly formed bone and bone marrow. (B, B´) D7 showed UBB stained region near
to periosteal region and bone marrow. (C) Active osteocyte signals visualized via Silver Nitrate
counter-stain within GPX1 positive areas. (D) Positive signal of UBB visualized around blood vessels
in the fractured callus via Silver Nitrate counter-stain. (E) GPX1 stained area was higher at D28
compared with other time points. (F) UBB stained area was significantly higher at D7, D10, and D14
when compared with D21. (N = 4 (D7), N = 6 (D10), N = 3 (D14), N = 5 (D21, D28), Ot: osteocytes).
These results encouraged us to investigate the major fusion genes of mammalian
mitochondria (Mfn1 and Mfn2; Mfn is mitofusin). Intriguingly both genes were down
regulated at all time points in the inflammatory and reparative phase and started to
upregulate by the beginning of the remodeling phase after bony consolidation at D21 and
D28, where the healing process is close to the end (Figure 2).
Figure 1: Mitochondrial fusion throughout the healing process in mouse model. The
expression of the major mitochondria fusion genes Mfn1 (A) and Mfn2 (B). Both genes were
downregulated from D3 to D14 when compared with D0. Upregulation of fusion genes came later
after the bony consolidation.
Nonetheless, quantification of mitochondria in histological sections is very important.
Histological samples are fixed, processed, embedded, and sectioned before being stained;
depending on these steps, the outcome of the study is affected. Cells are easily damaged
during the long steps of chemical fixation, once analysis under Transmission electron
microscope is envisaged. Therefore, we optimized the embedding process by the use of
microwave assisted chemical fixation using a PELCO BioWave® Pro microwave (Figure 3).
The low energy irradiation produced by the microwave during the fixation, dehydration and
resin infiltration enhances the diffusion throughout the samples and improved the
preservation quality tremendously [26]. These results showed that microwave technique
increases the quality of sample, preserves and enhances ultrastructural microscopy
analysis and the samples can also be used for further staining.
Figure 3: Microwave Assisted Chemical Fixation (MWCF) improves sample quality. (A) MWCF
is enhanced due to radiation, cooling plus vacuum and was used to fix samples. (B) Inferior
subcellular structure of bone samples in chemical fixation, Osteocyte with less recognizable
subcellular structure. (C) Enhanced ultrastructure in muscle cells. (D) MWCF fixation enhances
mitochondrial visualization of osteocyte.
Planned methodology:
Immediately
after
approval of the funding,
the experiments can be
started, since a large
part of the required
samples
is
already
available.
Samples
originate from volunteers
who visited UKGM for
fracture treatment or
cruciate ligament graft
will be analyzed (Figure
4). The ethics application
is already approved.
Other patient samples
will
be
additionally
acquired
until
the
required
number
of
Figure 4: Schematic illustration of the sample processing plan. samples is reached.
Starting with isolated MSCs with pro-inflammatory stimulation (red
arrows), anti-inflammatory stimulation (green arrows) and applied
strontium (yellow arrows).
Work Package I: Cell seeding, cultivation until confluency and cell profiling
Mesenchymal stroma cells (MSCs) will be used in the present study. MSCs are not only
able to differentiate into mesenchymal cells, such as osteoblasts, adipocytes and
chondrocytes, but also into non-mesenchymal cells including endothelial cells and neural
cells [27].
Cells will be transferred to expansion medium (DMEM, 10% FCS and 1% penicillin /
streptomycin). They will be incubated at 37°C in a CO2 incubator to allow them to attach to
the plastic flask. Cell splitting will take place as they became confluent in a 75-ml culture
flask.
Cell profiling will be performed using FACS Canto and the following markers CD13, CD45,
CD73, CD105, CD166 to confirm that the isolated cells are MSCs.
Work Package II: Cell stimulation and differentiation
Firstly, we are planning to induce a pro- respectively ant-inflammatory state of the isolated
MSCs.
Inflammation will be induced with tumor necrosis factor (TNF)-α (10 ng/ml) 1 h before the
experiment. Mitochondrial fission inhibitors Mdivi-1 or Dynasore will be added to cell culture
30 min prior to addition of TNF-α in the indicated experiments. Cytokines, growth factors
and extracellular matrix (ECM) proteins will be present due to inflammation. These in turn
stimulate proliferation of cells and healing [28; 29]. For example, Interleukin-1 (IL-1), an
inflammation producer which is mainly secreted by macrophages in the innate immune
response; Interleukin-6 (IL-6) an immune response stimulant which is secreted by T cells in
the adaptive immune response; both are known to recruit mesenchymal cells [29].
Effects of non-steroidal anti-inflammatory drugs (NSAIDs):
Pountos et al. [30] studied the effect of NSAID on MSCs proliferation and osteogenic and
chondrogenic differentiation and evaluated both cyclooxygenase (COX)-1 and COX-2
specific drugs. They tested the effects of seven COX-1 and COX-2 inhibitors on MSC
proliferation and osteogenic and chondrogenic differentiation. The MSC expression of COX1 and COX-2 and prostaglandin E2 (PGE-2) levels were evaluated by PCR and ELISA.
They found that none of the NSAIDs significantly affected MSC proliferation. Only MSC
chondrogenic was affected but not osteogenic differentiation [30].
To investigate the effect of NSAIDs on mitochondria fission. One of NSAIDs will be added
to MSCs culture with and without fission inhibitors to study the effect on proliferation and
osteogenic chondrogenic differentiation. Proliferation (using fluorescent dye or XTT assay,
to measure the number of viable cells), osteogenic (measure the activity of alkaline
phosphatase, ALP) and chondrogenic (measure the content of sGAG) differentiation will be
analyzed. Prostaglandin E2 (PGE-2) will be measured in the media and compare it with
cells without NSAIDs to check the activity of endogenous COX-1 and COX-2.
Effect of strontium on aging process in bone regeneration and mitochondrial dynamics
Because strontium has a stimulating effect on osteoblast and reduces osteoclast activity as
well as a positive effect on the mitochondrial membrane structure, we want to evaluate the
effect of strontium on isolated MSCs. Therefore, we will add 5%-10% strontium to the cell
culture medium of MSCs from young and old patients. The strontium treated MSCs will not
undergo a pro- or anti-inflammatory stimulation, but the osteo- respectively chondogenic
differentiation will be performed as described in the next paragraph.
Osteogenic differentiation assay
Differentiation will be performed in 24 well plates in triplicates with a control. Starter cell
density will be 2.4 x 104 cells in a 400 µl total volume of expansion medium per well, and
cells will be allowed to adhere for 48 hours. The first day of adding the differentiation
medium is considered day one. Osteogenic differentiation medium (ascorbic acid
phosphate, β-glycerophosphate, water-soluble Dexamethasone) will be changed twice
weekly. To test cell viability the 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) test will be used. Osteogenic differentiation will be
then evaluated visually by Von Kossa staining. Thereby, silver ions (one of the stain
components) bind to the phosphate adsorbed in the extracellular matrix and form silver
phosphate; which degrades to silver under light illumination. Calcium is measured by
alizarin red, the stain is then extracted (bleached) using cetylpyridinium chloride.
Subsequently dissolved color is measured at a wavelength of 405 nm by a microtiter platereader (TECAN, Genius – Maennedorf, Germany).
Chondrogenic differentiation assay
One million MSCs at passage 2 will be centrifuged at 200 g for 5 min and the pellets will be
cultured in 15 ml conical tubes containing 1 ml of chondrogenic differentiation medium
consisting of DMEM, 1× ITS, 100 nM dexamethasone, 50 mg/l ascorbic acid, and 5% PL.
The medium will be changed twice a week. After 3 weeks, chondrogenic differentiation will
be assessed via histochemical staining and RT-PCR. Pellets maintained in DMEM with 5%
PL will be used as negative controls [31]. Under the described culture conditions, human
MSCs undergo chondrogenic differentiation within 2–3 weeks, producing abundant
extracellular matrix composed primarily of cartilage-specific molecules such as type II
collagen and aggrecan. The expression of these cartilage markers can be used as evidence
of the chondrogenic differentiation of MSCs. Chondrogenic differentiation can be assessed
by toluidine blue staining of pellet sections.
Effect of Mdivi-1 on mitochondrial fission as inflammatory signal.
There are contradictory results regarding the use of Mdivi-1 to inhibit Drp-1 and prevent
mitochondrial fission [32]. The review of Smith and Gallo (2017) stressed on further
biochemical investigation and the use of stringent positive controls to proof the effect of
Mdivi-1 on fission.
Work Package III: Histology and TEM
Following the MSC stimulation and differentiation, histological examination will be
performed to verify the regenerative state of bone and mitochondrial state as a reaction to
the pro- and anti-inflammatory treatment. Therefore, the cells will be transferred onto glass
slides using the Cytospin system:
MSCs will be trypsinized and collected in 15 ml conical tubes. Cell number will be
determined, centrifuged and resuspended in PBS at a concentration of 5 x 105/ml.
Subsequently, 200 µl of cell suspension will be centrifuged on glass microscope slides using
a cytospin centrifuge. Afterwards, the cells will be fixed with 4% paraformaldehyde in NaPo 4
buffer and dried overnight to be ready for histological staining [33].
Immunohistology
Immunohistochlogy will be performed to detect osteo-anabolic (BMP2, OCN) and osteocatabolic markers (OPG, RANKL). Chondrogenic differentiation can be evaluated using
Immunohistochemical staining of serial sections of pellet sections such as Type I collagen,
type II collagen, type X collagen, TNF-a, DRP1, Fis1, Mfn1 and Mfn2.
Transmission Electron Microscopy (TEM)
For the TEM analysis samples will be collected directly and fixed in 2% (wt/v)
paraformaldehyde and 2% (w/v) glutaraldehyde in 0.05 M cacodylate buffer in a laboratory
microwave assisted chemical fixation (Ted Pella, Inc, Model PELCO BioWave® 34700230). Fixation will be done in four times under continuous vacuum of 15 mm Hg for 8 min,
a radiation power of 150 Wattage will be alternately applied between the fixation steps.
Then samples are washed with 50 mM cacodylate buffer four times using radiation power
of 150 Wattage for 2 min each without vacuum. After washing samples are dehydrated in
ascending series of ethanol: 30, 40, 50, 75, 90, 100% (vol/vol) ethanol in ddH2O with
radiation power of 15 mm Hg for 2 min each then in propylene oxide for 2 min with radiation
power of 15 mm Hg without vacuum. Sample infiltrate in ascending series of Spurr’s resin
mixed with propylene oxide takes place before embedding in 100% Spurr’s resin for 24
hours. The process will achieve fixation and embedding in 36 hours therefore, saving 97%
of the 6-8 weeks long embedded methyl methacrylate.
Work Package IV: Molecular biology
Molecular analysis will be performed to determine the bone regenerative capacity as well
as the mitochondrial state as a response to the different treatments. Therefore, we will use
different technics like PCRs and NGS.
qRT-PCR:
RNA will be isolated from cultured MSCs of all groups under different treatments and
treatments combinations. It will be used as templet for cDNA synthesis. Quantitative realtime PCR will be used for expression analysis. The following genes will be analyzed:
Anti-inflammation genes: COX-1, COX-2, to check if there is chondrogenesis
inhibition.
Anti-inflammation genes: IL-6, IL-8 to check the effect of TNF-α.
Transcription factors: Runx2 and Sox-9 triggers of osteogenesis and
chondrogenesis, respectively.
Mitochondrial fission genes: Fis1 (promoter of mitochondrial fission), DRP1 (fission
process). Under inflammation should be increased
Mitochondrial fusion genes: OPA1, MFN1, MFN2. Under inflammation should be
decreased.
Effect Wnt signaling pathway: dKK, Sost
House-keeping gene: GAPDH
NGS:
RNA isolation will be performed using NGS Tool Kit, Takara SMARTer® Ultra® Low Input
RNA for Illumina® Sequencing – HV according to manufacturer’s protocol.
Next generation sequencing will be performed at the university core facility after applying
the prepared RNA on the NGS-chip according to manufacturer’s protocol.
The NGS data are then analyzed using bioinformatics tools with focus on the coding
components. The analysis will run using R packages such as Bioconductor for NGS data
analysis. The main goal is to form enrichment maps to understand network analysis
between the genes associated in bone, fat metabolism and mitochondria.
ELISA: (collaboration)
Concentrations of interleukin-6 (IL-6) and -8 (IL-8) in cell culture supernatants will be
measured by ELISA. To correlate the presence of TNF-α with the level of IL-6 and IL-8 in
presence and absence of strontium.
Microarray, protein array and western blot: (collaboration)
Microarray: The human mitochondrial genome is circular with 16,569 base pairs and
encodes 37 genes coding for 22 transport RNAs, 2 ribosomal RNAs, and 13 messenger
RNAs [34]. Microarray is used to study alterations of mitochondrial and nuclear gene
expression in healthy and diseases samples. Commercially available mitochondrial array
will be used.
Protein array: Proteins will be extracted from different treatments with and without
inflammatory conductions and will be applied on available protein arrays i.e. stress-related
proteins and apoptosis-related proteins.
Western blot: WB will be performed using specific antibodies to check the expression or
repression for FIS1, DRP1, MFN2 and β-actin (house-keeping protein): with/without TNFα; with/without strontium; with/without Mdivi1.
Work Package V: Bioinformatics
Quantitative immunohistological evaluation will be done semi-automated using ImageJ
software (1,52k) to quantify the mitochondrial and regenerative state after treatment and
differentiation.
The NGS raw data will be analyzed using the R project for Statistical Computing. Quality
assessment of array data will be carried out by calculating Pearson’s correlation coefficient
between arrays and fold Change (FC) will be calculated as the differences between each
treatment. Clustering of genes will base on the expression pattern identifies co-regulated
and functionally related genes. Hierarchical clustering method using R (version 3.0.3) and
Bioconductor packages will be implemented to obtain set of differentially expressed genes
with similar expression profile. An online server; Database for Annotation, Visualization and
Integrated
Discovery
(DAVID)
bioinformatics
database
(version
6.7,
https://david.ncifcrf.gov/) provides an integrated and updated information about the involved
molecular function, cellular component, and biological processes for gene of interest.
Furthermore, DAVID provides an option of functional annotation clustering to cluster
functionally similar terms associated with gene list.
Conclusion
The aim of this research proposal is to unravel the mechanisms underlying modulation of
endogenous regeneration due to aging, with a focus on mitochondrial network dynamic in vitro.
Special attention is given to the response of mesenchymal stromal cells to pro- and antiinflammatory stimulants and in response to prophylactic drugs.
Publications
1.
El Khassawna T, Serra A, Bucher CH, Petersen A, Schlundt C, Könnecke I,
Malhan D, Wendler S, Schell H, Volk H-D, Schmidt-Bleek K, Duda GN: T Lymphocytes
Influence the Mineralization Process of Bone. Front Immunol 2017, 8. [doi:
10.3389/fimmu.2017.00562]
2.
El Khassawna T, Bocker W, Brodsky K, Weisweiler D, Govindarajan P,
Kampschulte M, Thormann U, Henss A, Rohnke M, Bauer N, Muller R, Deutsch A,
Ignatius A, Durselen L, Langheinrich A, Lips KS, Schnettler R, Heiss C. Impaired
extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.
Histochem Cell Biol. 2015 [doi: 10.1007/s00418-015-1356-9]
3.
Schlundt C*, El Khassawna T*, Serra A, Dienelt A, Wendler S, Schell H, van
Rooijen N, Radbruch A, Lucius R, Hartmann S, Duda GN, Schmidt-Bleek K. Macrophages
in bone fracture healing: Their essential role in endochondral ossification. Bone. 2015.
(Equal contribution) doi: 10.1016/j.bone.2015.10.019.]
4.
El Khassawna T, Bocker W, Govindarajan P, Schliefke N, Hurter B, Kampschulte
M, Schlewitz G, Alt V, Lips KS, Faulenbach M, Mollmann H, Zahner D, Durselen L,
Ignatius A, Bauer N, Wenisch S, Langheinrich AC, Schnettler R, Heiss C. Effects of
multideficiencies-diet on bone parameters of peripheral bone in ovariectomized mature
rat. PLoS One. 2013. [doi: https://doi.org/10.1371/journal.pone.0071665]
5.
El Khassawna T. Cellular and molecular analysis of fracture healing in a
neurofibromatosis type 1 conditional knockout mice model
[https://doi.org/10.18452/16781]
Collaboration with other scholars:
Prof. Klaus D Jandt
Prof. Dr.-Ing. Peter Czermak
Univ.-Prof. Dr. med. Christian Heiß
Lehrstuhl für Materialwissenschaft, Otto-Schott-Institut für
Materialforschung, Friedrich-Schiller-Universität Jena
Technische Hochschule Mittelhessen
Prof. Dr. Katrin Susanne Lips
Univ. Prof. Dr. Ing. Georg Duda
Klinik
und
Poliklinik
für
Unfall-,
Handund
Wiederherstellungschirurgie,
Justus-Liebig-Universität
Gießen
Klinik
und
Poliklinik
für
Unfall-,
Handund
Wiederherstellungschirurgie,
Justus-Liebig-Universität
Gießen
Justus Liebig Universität Gießen
Julius Wolff Instiut, Universitätmedizin Charite, Berlin
Prof. Kurosch Rezwan
University Bremen, Department 4, Advanced Ceramics
Prof. Michael Gelinsky
Zentrum für Translationale Knochen-,
Gelenk- und Weichgewebeforschung,
TU Dresden
Klinik und Poliklinik für Kinder- und Jugendmedizin,
TU Dresden
Physikalisch-Chemisches Institut, JLU Gießen
Leibniz University Hannover,
Institute for
Multiphase Processes
Medizinische Klinik V, Labor für Myelomforschung,
UK Heidelberg
Prof. Dr.med. Volker Alt
Prof. Angela Rösen-Wolff
Prof. Jürgen Janek
Prof. Birgit Glasmacher
Dr. Dirk Hose
References:
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