Summary of Doctoral thesis: Experimental research to apply protocol culture and cryopreservation of osteoblast from differentiation bone marrow mesenchymal stem cell

Isolation, identification and culture successfully mesenchymal stem cell sample from rabbit bone marrow. Differentation mesenchymal stem cell towards the osteoblast and cryopreservation cell after differentiation.

4.2.3.Assessment of cell viability

Most of the thawed cells can attach surface of culture flasks In

addition to characteristics described above, another defining feature that

thawed osteoblast still kept was their morphology These observations

clearly showed cryopreserved osteoblast could be stored and maintained

hight degrees of viability and differentiation potential With these

results, we believed that cryopreserved osteoblast might become

promising candidate cell source for cell –based therapy

CONCLUSIONS

1 Isolation, identify and culture successfully mesenchymal stem cells

from the rabbit bone marrow, thereby providing a summary process

for important steps such as anesthesia, position, aspirated marrow

technology, isolation, culture mesenchymal stem cell (identified as

mesenchymal stem cell)

2 Differentiation successful bone marrow mesenchymal stem cell into

osteoblast After cell differentiation were identified by

immunohistochemistry with antibody osteocalcin, staining with

Alizarin red, observation of mineral crystal under scanning electron

microscope The result confirm the differentiation mesenchymal

stem cell into osteoblast Cryopreservation cell after differentiation

by slow freezing, rapid thawing in freezing medium 10% DMSO,

15% FBS After thawing, viability rate over 80% Most of the

thawed cells can attach surface of culture flasks, cell is still

developing well, cell shape is unchanged

RECOMMENDATIONS

1 Optimal protocol of isolation, culture, differentiation mesenchymal

stem cells towards osteoblast from human bone marrow

2 Apply autologous grafting mesenchymal stem cell differentiation

osteoblast for patient, who graft bone tissue and bone disease

BACKGROUND Partial loss of a bone of the extremities due to trauma, tumor excision, bone disease, etc… presents as a substantial impediment throught the life of patients, and numerous efforts have been made to reconstruct the normal function of the skeletal system However, such efforts still have many limitations and problems When using bone graft, problems may develop in the donor area in general autologous bone graft and immunological problems while the spread of disease may also develop in allografts

Despite advanced and optimized surgical procedures, approximately 5% fracture sustained annually in the United states fail to achieve bony union There may be faster patient recovery and an absence

of these problems when stem cell are used Cell culture has large numbers Cell differtiation, they would be expected to become a useful cell source for treatment, has become more research favorable in the world and Vietnames Therefore, the maintenance of stem cell initial characteristics is crucial, and it may be possible to preserve them by satisfactory cryopreservation technologies This is expeccially significant because the supply of stem cells that are limited could be happed any time

In fact, if long term cryopreserved stem cell still retained biological ability, they would be expected to become a useful source for regenerative medical progression

Everyday, our laboratories we provide bone tissue for 10 patient to autograft and allograft Purpose bone tissue engineering conjugate with stem cell engineering that can regenerate bone tissue with an appropriate structure and function Therefore, we conducted “experimental research

to apply protocol culture and cryopreservation of osteoblast from differentiation bone marrow mesenchymal stem cell”

Objective:

1 Isolation, identification and culture successfully mesenchymal stem cell sample from rabbit bone marrow

2 Differentation mesenchymal stem cell towards the osteoblast and cryopreservation cell after differentiation

The thesis is a new contribution to the field of stem cell research in

Vietnam Especially successful differentiation of bone marrow

mesenchymal stem cell into osteoblast with morder identify technology

This is the first result in Vietnam This result has great practical

significance in the treatment of bone and joint disease by stem cell therapy

Through the thesis, the author and the stem cell research team of

the Department of Histology and Embryology, Ha noi Medical

University have mastered technology as the steps in a process, starting

with the isolation technique, culture, differentiation, identify,

cryopreservation cell We will build a cell bank that is differentiation

according to treatment and research

STRUCTURE OF THE THESIS The thesis consitts of 127 pages, 4 chapters, 11 tables, 45 figures,

references: 9 Vietnamese: 116 foreign language

Background: 02 pages; chapter1: overview 37 pages; chapter 2:

Objects and Methods:18 pages; Chapter 3: results: 30 pages; Chapter 4:

Discussion 37 pages; conclusions: 02 page Recommendations: 01 page;

lists of related articles: references; appendix

CHAPTER 1: OVERVIEW 1.1 Mensenchymal stem cell

1.1.1 Character mesenchymal stem cell

Friedenstein AJ (1976) were discovered stromal stem cell, Who

showed that bone marrow contains a population of plastic- adherent

highly proliferative cells, that were able to form colony of fibroblast

(hence the name colony – forming unit – fibroblast, CFU-F)

Mesenchymay stem cell (MSC) display multilineage differentiation

potentials Invitro, MSC are capable of differentiation a long

osteogenic, chondrogenic and adipogenic lineages

Morphological MSC population are fusiform or stellate cell not

easily distinguished from fibroblast In electron micrographs, they have

a coarser chromatin pattern, fewer mitochondria and their sparse

cytoplasm contains little or no endoplasmic reticulum

4.1.3.8 Injection of autologous cultured osteoblast in experimental

When we compare the results in the two groups studied the results showed that in the experimental, the time for new bone tissue regeneration and healing of bone quality was better and faster the control group at 3 week There results were demonstrated histologic image Afetr 6 weeks, the comparisons between the team were very difficult However, if based on SEM picture, the regeneration and repair in experimental were not similar, while the team for bone healing pretty good results, the results one the bone healing not as good as the control group

4.2.Cryopreserved osteoblast

4.2.1.Cell viability rate

The slow freezing – rapid thawing method using dimethylsulfoxide (DMSO) as a cryoprotectant can be easly applied to the cryopreservation of cell such as osteoblast The cell are suspended in freezing medium containing 10% DMSO transferred into cryovials and then frozen by steps with slowly decreasing temperatures 40C for 10 min, -200C for 1 hour, -800C for 1-2 days and -1960C for 2 weeks In addition to keeping balance to protect cryopreservated cells, toxic and osmotic damage by decreasing temperature slowly with low concentration of cryoprotectants When slow freezing method cryoprotectant as replacement for intracellular water The viability of osteoblast thawed from different freezing media varied as well However the difference between viability rate of cryopreserved groups and freezing medium After thawing, cryopreserved cells had a fairly hight degree of viability, about 87% in MT3 freezing medium MT1 freezing medium was not suitable, the viability rates had low 61.28% Our study was suitable result of Verdanova M (2014) Cryopreserved osteoblast in differentce freezing medium the cells were frozen in freezing tubes at -800C at a cooling rate of 10C/min Frozen cells were transferred into liquid nitrogen 87% of frozen osteoblast survived 24h after thawing from the standard freezing medium 10% DMSO and 25% FBS

The osteogenic potential of the isolated mesenchymal cells was

confirmed with the qualitative analysis based on the staining of the

calcium accumulated in the extracellular matrix, which is one of the

main events in the osteogenic differentiation process The evaluation

time points 7-14 days the cell under osteogenic conditions exhibited

morphological changes typical of the osteoblastic phenotype The

observation of the major mineralization events after 30 day of culture is

white crystals in accordance with previous reports for MSCs suggesting

similar osteogenic potentials(Quiroz FG 2008)

MSC were plated and treated for osteogenic differentiation as

described To assess differentiation of rabbit bone marrow

mesenchymal stem cells, some bone – specific markers were analyzed

using histo and immunohisto- chemistry techniques in cells grown in

osteogenic differentiation medium There aggregates were characterized

by deposits of amorphous material The nodular aggregates in

osteogenic cultures stained with alizarin red S, demonstrated that

amorphous deposits observed under the microscope were actual calcium

deposits Alizarin red – positive nodular aggregated were already

present at day 15 The positive alizarin red were lager and stained

intensively, indicating that a more extensive calcium deposition had

occurred Control cultures showed only minimal background staining

Under SEM appearance white crystals of mesenchymal stem cells

differentiation into osteogenic line The cells were

immunocytochemistry stained positive for osteocalcin Osteocalcin, a

bone – specific glycoprotein that binds calcium and may promote

calcification of the bone matrix has been used as a late marker for

osteogenic differtiation (Karaoz, 2009)

The bone marrow mesenchymal stem cells model used in this study

has basal expression of the osteogenic markers, only cultures under

osteogenic conditions showed osteocalcin marker for the induction of

the osteogenic differentiation as evidenced though the morphological

changes and the mineralization of the extracellular matrix

However, MSC constitute only a small proportion of the cell in bone marrow 0,001 – 0,01% of total nucleated cells ̴ 1/10 HSC Now, MSC were isolated from various sourcer as umbilical cord blood, adipose tissue, bone marrow, cartilage…

1.1.2 Marker MSC (identified by the minimal criteria of the

international society for cellular therapy

Table 1: Marker for human mensenchymal stem cell

Positive ≥ 95% Negative ≤ 2%

CD105, CD73, CD90 CD45, CD34, CD14 or CD11b,

CD79α or CD19, HLA-DR

However, this study investigated whether there are marked differences in surface markers between rabbit and human MSC

1.1.3.BMMSC isolation and detection

BMMSC were isolated from the marrow aspirate by gradient centrifugation, using Ficoll –Paque (Sigma, USA) The mononuclear on the top with an equal volume of Ficoll – Paque

The isolated cells contains MSC, hematopoitic cells and monocytes The culture remove the hematopoietic cells and futher expanded MSC

1.2 Criteria to define MSC (ISCT) One notable characteristic is plastic – adherent ability of MSCs ,When they are cultured in appropriate media Another property MSCs must have differentiation potential into osteoblasts, adipocytes and chondroblasts in ni vitro culture with know stimulators Lastly, MSCs were confirmed with the phenotype that were positive for CD73, CD90, CD105 and negative for CD45, CD34, CD14 or CD11b, CD79α

or CD19 and HLA-DR

1.3 In vitro differentiation:

MSCs are multipotent cells that can undergo multi –lineage differentiation MSCs were differentiation in to osteoblast, adipocytes, chondroblasts… using the standard induction media

1.4 Cryopreservation cells

Cryopreservation cells sequentially stored with slowly decreasing

temperatures Between -50C to approximately -150C, ice forms (either

spontaneously or as result of artificially introducing ice crystals to the

solution) in the external medium; however, the cell’s contents remain

unfrozen As the cell increased solute concentrate and cryoprotectants

(CPAs) in response to increasing extracellular osmolality It is clear from

the cryobiology theory decribed above that whether the ability of water to

move across the cell membrane agiven cell type depends cooling rate

The subsequent physical events in the cells depend on the cooling

rate If cells are cooled enough slowly intracellular solution maintain

equilibrium with external medium However, if cells are cooled too

rapidly than the water transport external medium as result introducing

ice crystals intracellular cell, but morphological cells aren’t change

Following storage, optimal cooling cell will lose water in the

partialy enough to maintain equilibrium

If the cell aren’t cooled rapidly, ice crystals froms in the extermal

medium, they will experience a severe volume shrinkage

In general, cryopreserved cells are equilibrium between losen water

and cooling rate

Role cryoprotectants

All discussion above refer to the role of CPAs helps avoid the risk

and possible damage caused by low temperature or frozen temperature

CPAs is induce the cell water/cytoplasm to form a glass than to

crystallize The CPAs suppress the salt concentration in solution; reduce

harmful cell shrinkage at any given temperature In the presence of

macromolecular compounds, a proportion of water does not crystallize

during the freezing process

Cryopresesation stem cell

Xie XH (2012) Cryopreserved rabbit bone marrow mononucle

approximately 107 cell/1ml re – suspended cryopreserved medium 10%

expressed CD44, CD90 They also were negative for CD14,CD34 These results demonstrated that bone marrow MSCs, in this study, is relatively satisfactory to recommended minimal criteria of the mesenchymal and tissue sem cel committee of the international society for cellular therapy CD14, CD34 are common markers of circulating blood cells Among of them, marker Cd14 lipo- polysaccharide receptor, is a surface antigen of monocytes, macrophages and endothelial progenitor cells The CD34 protein, a surface glycoprotein, expressed on developmentally early hematopoietic stem cells and progenitor cell as well as endothelial cells It has been suggested that CD34 negative stem cell may generate hematopoietic stem cell

Lee TC (2014) reported that the surface expression of 9 markers differed between human and rabbit MSCs, Whereas the surface expression of 16 markers was he same in the two cell lines Analysis results also showed that rMSC were positive with markers CD44 (97.32%), CD90 (95.37%) and the rate of negative markers are CD14 (0.17%), CD34 (0.36%)

4.1.3.6 Adipogenic differentiation

rMSC in the adipogenic differentiation experiments Although adipogenic differentiation potency of rabbit bone marrow MSC was examined for 10-15 days, the onset of phenotypic changes was noticed after 5 -7 days of incubation During differentiation, cell shape began to transform into ovoid morphology and the lipid droplets showed a tendency to accumulate in the cell periphery At the beginning of the second week, the lipid droplets enlarged and invaded the entire cytoplasm like adipocyte differentiated rabbit bone marrow MSCs Control cells did not differentiate Oil red O staining positive adipocytes were red color which demonstrated the committed differentiation of MSC into adipocytes when culture in induced medium with know adipogenic factors

4.1.3.7.Osteogenic differentiation

marrow graft obtained after concentration cantained progenitor cells

The use of percutaneous autologous bone marrow transplantation in

nounion and a vascular necrosis of bone

4.1.3.4.Characterization of cell proliferation assay

The proliferation of the cell lines was demonstrated by growth

curves for the measurement of cell proliferation the growth curves test

was performed The cell proliferation was using evaluation method

effects of growth factor, hormone, nutrient medium, difference factor

on cell line The cell growth or increases the number of cell is that work

has been carried out on almost all aspects of mesenchymal stem cell

biology Because they were determined effect difference factors:

altering nutrition, mitosis factor…

Liu C (2014) reported that Annulus fibrosus (AF) sample were

isolated from intervertebral disc (IVD) of rabbits The cell was cultured

in DMEM – LG supplemented with 20% FBS, 100 U/ml penicillin, 100

µg/ml streptomycin Cell proliferation capacity was tested using cells at

passage three though MTT assay The typical population doubling time

was 17.8 hrs

4.1.3.5 Expression of surface markers

Immunofluoresvence staining and flow cytometry analysis

indicated that MSC at P4 were strongly positive CD44,CD90 The cells

were negative for CD14, CD34 All markers are commonly used in

analyses of MSCs These data coincide well with the reported data of

mesenchymal stem cells characteristics

Tan SL (2013) repored that both rMSC and hMSC at P2 or P3 were

determined for phenotypic characterization using flow cytometry The

cultured rMSC were positive CD90 (96,9%) and hMSC (100%) Our

result were positive CD90 (95.37%)

Lee TC (2014) studied that rMSC CD44 (97.32%) This result was

suitable with studied of ours

Characterization of bone marrow mesenchymal stem cells is also

based on the expression of specific markers MSCs were strongly

DMSO and 90% FBS After storage for 8 weeks, cryopreserved cells had a fairly high degree of viability, about 96,49%

Kotobuki N (2005) cryopreserved MSCs in medium: DMSO and FBS After storage for (0,3-37 months), the viability rates of cryopreserved groups were approximately 90% Both fresh and cryopreserved MSCs analysis their marker This result also revealed that the cryopresenvation did not influence the marker

Zeisberger SM (2011) cryopreserved AT- MSC for a concentration

106 cell/ml Cryovials were frozen and sequentially stored with cooling rate 10C/min to -800C for 2 days Immediately after wards, the cryovials were immersed in liquid nitrogen

Following storage, cryopreserved cells were medium with diffence concentrate, DMSO will difference ice froms intracellular and extracellular solution

Shimizu T (2013) cryopreserved osteogenic matrix cell sheets (OMCS), in which bone marrow - derived MSC were culture to confluence and differentiated the osteogenic lineage to form osteogenic matrix cell sheets (OMCS) OMCS are into cryovials containing cryopreservation medium (cell Banker 1®) Tube were then transferred

to a freezer (-800C) and stored at -800C for 4 or 12 weeks After thawing, the viability rate of cryopreserved groups were 63,3± 8.6%; 61.1 ± 6.5%, respectively After thawed OMSC are still capable of producing minerlzed There was no significant difference in ALP activing between the fresh, 4 week and 12 week groups

CHAPTER 2 SUBJECTS AND METHODS 2.1 Subjects of the reseach

 Subjects reseach: Bone marrow mononuclear cells were isolated from the marrow

 Material reseach: Bone marrow solution

 Standard of rabbit: 30 rabbit, 6-8 week old male rabbit for 1.8-2.5kg

 Total sample bone marrow :30 sample, culture 30 sample,

cryopreservation 30 sample

2.2 Methods of the reseach

2.2.1 Reseach model

 Isolation, culture, identified and differentiation MSC from bone

marrow

 Cryopreservation osteoblast then evaluation of viability rate and

differentiation ability of cryopreserved groups

2.2.2 Content reaeach

2.2.2.1 Isolation, culture, identified MSC from BM and differentiation

into osteoblast

Aspirated BM: Choice of position bone marrow aspirate and total

volume solution

Isolationed MSC: Bone marrow mononuclear cells by gradient

centrifugation Briefly, bone marrow was loaded on the top with an

equal volume of Ficoll –Paque (sigma, USA) preloaded in a centrifuge

tube and centrifuged at 3000rpm for 20 minutes in room temperature

Culture:

Primary culture: Bone marrow mononuclear cells were suspended

in culture medium and seeded into flask The cells were cultured at

370C, 5% C02 The subsequent medium change was conducted at 3 day

intervals When these primary culture cells reached 70 – 80%

confluence, they were sub cultured

Second culture: These primary culture cells were harvested using

Trypsin/EDTA and sub cultured at 1: 3

Detection cell:

Immunostaining cells:

Cells were fixed with 4% PFA at room temperature for 10 min and

washed with PBS, followed by permeabilization with 0,2% Triton

X-100 for 10 min and blockage with 4% goat serum Cells were incubated

with primary for 1 day 250C Subsequently, antibodies of CD14, CD34,

Under the culture condition used in this study, rBm-MSC grew consistently for 10 Passages without loss of proliferative ability and significant changes in morphology

4.1.3.2 Second culture

The cells, referred to stem cells, proliferated more rapidly than primary culture (in the first passage of culture), fibroblast – like cells reached 70 – 80% confluence at day 5 -7, at which point these cells were suitable sub cultured These cells were detached by incubating with trypsin/EDTA solution Complete medium was added to inactivate the trypsin and sub cultured at a ratio of 1:3 Purification of mensenchymal stem cells candidates was achieved by removing nonadherent cells during sub sequent changes of medium When sub culture homogenous after passage three

4.1.3.3 Conony formation

Such stem cells should possess clonogenicity, self – renewal capability and multipotency the common characteristics of mesenchymal stem cell

To test the colony forming capability of mesenchymal stem cell, a colony forming unit – fibroblast (CFU-F) assay was performed Base on size, morphology of cell was knew progenitor cells Our study MSCs were culture in the growth medium supplemented with 10% FBS The number of fibroblast colony –forming mean 107 ± 25

Liu C (2014) to test the colony forming capability of rabbit AF – derived cells, they were cutured in the growth medium supplemented with 20% FBS The colony formation capacity was largely dependent

on the initial cell seeding density An initial seeding of density of 200 cells/cm2 was found to result in the highest efficiency of colony formation

To test the colony forming unit (CFU-F) as an indicator of stromal cell activity Results are expressed as the mean number of mesenchymal stem cell per one million mononucleated This technique, the number of mesenchymal stem cell that was transplanted was estimated by counting the fibroblast conoly forming units (Hernigou.PH 2005) The bone

adherent, their shape and size and their ability to help form colonies in

culture Mensenchymal stem cell obtained by this method were

adherent to plastic and demonstrated fibroblastic spindle shape as

observed under microscope

In present study, spindle – shaped fibroblast – like cells from bone

marrow were purified by repeated medium chang at initial hours of

culture and diminishing the trypsinization time

Using the method described in this study frequent medium change

may prevent adherence of many of the non – MSC and hematopoietic

population to the culture dish

In the study, The mean number of rMSC was determined in

primary culture

Phase 1: The mean number of adherent cell and proliferation was

lower than Phase 2 because the first reseach haven’t stardard protocol

Secon reseach: The mean number MSC in P0 was 1.8

x104cell/106BMMNCs MSC candidate proliferated slowly in primary

culture Our results was suitable for XieXH (2012) MSC in P0 was

1.92 x 104/106 BMMNCs

Mesenchymal stem cells obtained by this method were relatively

homogeneous in morphology rMSC characteristics similar to those of

hMSCs based on the following observing under light microscope The

mesenchymal stem cell are small fusiform or stellate cells In electron

micrographs, they have a coarser chromatin pattern, fewer mitochondria

and their sparse cytoplasm contains little no endoplasmic reticulum

Although similar in their morphological appearance the rMSC cell

length (202.66 ± 840µm) and width (13.09 ±0.91µm) were significantly

longer and wider than hMSCs (152.04 ± 43.35µm) in length; 9.82

±0.66µm in width (p<0.01) (Tan SL etall, 2013)

Isolation of rMSC was achieved by using the Ficoll- paque gradient

centrifugation 30 samples were isolated and expanded in vitro This

technique stratified the MSC – like population cell into single layer

Rate of samples culture successful were 100%

CD44, CD90, and then conjugated secondary antibody staining solution for 30 min at 370C, cells were wash with PBS and DAPI staining

Flow cytometry:

The rabbit MSCs were characterized for the expression of surface marker of MSC using FACS analysis with antibodies of CD14, CD34, CD44, CD90

MSC display multilineage differentiation potentials:

Differentiation adipocytes

To induce adipocytes differentiation, MSC were seeded on the 6 well plates cells Cells were exposed to DMEM supplemented with 50µg indomethacin, 50 µg ascorbic acid, 10-7 dexamethasone for 2-3 weeks The medium was changed every 3 - 4 days Intracellular lipid droplets indicating adipogenic differtiation were observed under a light microscope and confirmed by Oil red O solution, as an indicator of intracellular lipid accumulation

Diffrentiation osteoblast:

To induce osteoblast differentiation of the MSCs, the culture were maintained in osteogenic media supplement with 50 µg ascorbic acid, 10mM β - glycerol phosphate, 10-7M dexamethasone Medium was changed every 3- 4 days Osteogenic differentiation was evaluated at 21 post induction Alizarin red staining and SEM

Alizarin red S stain: Method was used to verify the state of

osteogenic differentiation in term of extracellular matrix minerlization Briefly, cells were fixed with 70% ethanol and then alizarinred 2%, pH4.0 were added for 5 minutes Alizarin red S solution was washed with water and cultures were evaluated microscope The results were analyzed qualitatively based on the intensity of the staining and the extension of the alizarin – stained positively areas (red spots) Matrix mineralization was evident in the form of calcium nodules (stained red)

in the cultures under osteogenic conditions

Immunocytochemisty staining: To confirm post differentiation

MSC can be assessed by differentiation into osteoblast, postive marker

of osteocalcin ?

SEM: White crystals appeared in osteoblast differentiation were

observed under SEM ?

Injection of autologous cultured osteoblast in experimental: A total

of 15 white rabbits were divided into group in control and experimental

group To aspirate bone marrow, isolation, culture, diffrentation MSC

into osteoblast as reseach protocol To bone defect: Radial shaft defects

size 0.5 x 0.5cm in the body of rabbit femur The culture cell prepare,

the number was 106/ml in one vial The cell contained in the syring

were injected into the defect area The rabbit studies followed at the

time points 3,6 weeks

2.2.2.2 Osteoblast cryopreservation

Post differentiation MSC into osteoblast for 10 days, they were

trypsinzed by 0.25% trypsin/ EDTA (sigma, USA) and centrifuged at

3000rpm for 5 minutes at room temperature to remove extra trypsin

Approximately 106- 107 cells/ml re- suspended cryopreserved medium

10% DMSO with 0% FBS, 15% FBS, 30% FBS, repectively Cryovials

were frozen and sequentially stored with slowly decreasing

temperatures, incubated at 40C for 10 minutes, cooled to -200C and then

cooled to -800C for 1 day Immediately after wards, the cryovials were

immersed in liquid nitrogen

Cell thawed

Following storage, cryopreserved cells were thawed by rapidly

immersing the cryovial in a water bath 370C All cell suspension were

resuspended in culture medium and centrifuged to collect cells at the

bottom of the centrifuge tube After thawing, cell number and the

viability of post – cryopreserved osteoblast was determinerd by trypan

blue stain

Survival of osteoblast after thawing

In order to examine the ability of osteoblast development and

morphological characteristics Thawed osteoblast were seeded into

flasks They cells were culture at 370C, % C02

Total marrow volume for 10 ml is suitable in isolation and cultivation Our result is suitable with Huang JW (2006), Xie XH (2012)

4.1.2 Volume and quality bone marrow

Phase 1: 17 marrow samples were aspirated from iliac crests The marrow is aspirated in small and be cotted ̴ 30% sample because The first reseach haven’t standard protocol

Phase 2: According to our experience with 30 rabbit with standard protocol Therefore, method capable of increasing the volume and total number of nucleated cells rather than on the concentration of cell in phase 1

Huang JW et all (2006) have evaluate for 10ml volume, Which is suitable enough cells in cultivation The marrow is aspirated in small amounts to reduce the degree of dilution by peripheral blood and a void

to effect health of rabbit

Our results showed that the number of bone marrow mononuclear isolation per ml of bone marrow was 6.6 ± 0.3.8 x 106 Xie XH (2012) have described the same reseach, Which aspirated for 10ml marrow per rabbit and the mean number of bone marrow mononuclear showed per

ml 6.2 ± 0.86 x 106

4.1.3 Cell culture, proliferation and colony forming unit of MSC 4.1.3.1 Primary Culture

In previous reports, the population of mesenchymal stem cell was knowm to difficult to isolate because they were usually contaminate by hematopoitic progenitors and stem cells In addition, other ideals were suggested that the cells were heterogeneous in morphology and surface molecules Is was emphasized the presence of differently shaped and sized cells The small one, referred to stem cell, proliferate more rapidly than the large one, seem to as mature mensenchymal stem cells

In this study, however, we indicated that a relatively homogeneous population rabbit bone marrow method involving in the biological characteristics of mensenchymal stem cell, including their tendency to

There was a positive correlation between the cell viability rate and

MT1, MT3 groups were positive correlation, correlation coefficient is r

= 0,316 The approximate straight line is y = 0.432X + 51.57

3.2.2 Culture osteoblast after thawing

To check whether cryopreserved osteoblast have development and

morphology comparable to those of noncryopreserved cells We

analyzed the cell culture of 3 groups were different freezing medium

The cell morphology and proliferation was determined by light

microscope, no significant difference was observed between different

cryopreserved groups and control groups

CHAPTER 4 DISCUSSIONS 4.1 Aspirated, Isolation, culture, identified and differentiation

MSC from bone marrow

4.1.1 Choice position aspirated marrow

Section position aspirated marrow is very important If marrow is

enough volume, cell should not enough number in cultivation

Marrow was aspirated in ischia tuberosity, greater trochanter of

femur position in 5 rabbit, repectively but there are not successful Iliac

crest position is suitable for rabbit marrow aspiration

Under sterile conditions a 5 mm skin incision is made at level of

the iliac crest The needle for bone marrow aspiration is then pushed by

hand about 8mm into the cancellous bone of the iliac crest so that the

tip lies between the inner and outer table If no marrow is obtained, the

needle should be reoriented within the ilium and the aspiration should

be repeated It may be necessary to change the trocar insertion site if no

marrow is obtained At a given depth where marrow is successful

aspirated, the troca is turned 300 during successive aspiration to reorient

the bevel, thereby affording aspiration from the largest possible space

The bone marrow is aspirated into a 3ml plastic syringe The marrow is

aspirated in small (0.5-1ml) amounts to reduce the degree of dilution by

peripheral blood All aspirates are pooled in falcon containing an anti

coagulant solution

CHAPTER 3: FINDINGS 3.1 Isolation, culture, identified and differentiation MSC from bone marrow

3.1.1 Results of studies volume and quality bone marrow

Under local anesthesia, 47 sample bone marrow suspension was harvested from posterior iliac crest Mononuclear cells were isolated bone marrow and cultivated

Table 1 Volume quality of harvested bone marrow

Phase research Phase 1

(n=17)

Phase 2 (n=30) Volume of harvested bone marrow, ml 7.5±3.1 12±4.3

No.of BMMNCs in bone marrow x 106 per ml of bone marrow

5.8±6.5 6.68±1.12

Note:

Phase 1: Bone marrow sample were cotting (30%), Number of bone marrow mononuclear cells was very low: 2.53±3.45

Phase 2: The mean volum of 30 sample of the collected bone marrow was ̴12 ml Thus, the mean number of bone marrow mononuclear ceels isolated per ml of bone marrow was determined (Table 1)

3.1.2 Resul of culture MSC 3.1.2.1 Resul of primary culture MSC

Phase 1: 17 isolated bone marrow samples, culture suscefful rate 23% Phase 2: 30 isolated bone marrow samples, culture suscefful rate 86.7% (4 culture samples after 15 days, these primary culture cells do not reached 70% - 80%)

Table 2 Number of primary MSC from bone marrow mononuclear cells

Phase research Phase 1 Phase 2 No.of BMMNCs in bone marrow x

106 per ml of bone marrow 5.8 ± 6.5 6.7 ± 1.12

No of adherent cell x 104/ No of

Note:

Phase 1:Number of adherent cell/ number bone marrow

mononuclear cells: 2.53 x104 cells

Phase 2: The mean number of adherent 30 samplewere 12.04 x104 cells

Morphological characteristics of MSCs

Bone marrow mononuclear cells were seeded with a mean density

of 6.7 x106 per flask After 48 hours medium changes, which resulted in

the removal of the majority if not all non – adherent cells, the remaining

cells appeared to have heterogeneous fibroblastic – like (3.2) In the

first passage of culture fibroblast – like cells reached 70-80%

confluence at day 10-12 days (3.3)

MSC candidates were

cultured after 5 days (x200)

MSC candidates were cultured after 10 days (x200) Fig 3.1 In the first passage of culture, fibroblast – like cells

researched 70- 80% confluence at day 10

3.1.2.2 Second culture

When these primary culture cells reached nearly confluence, at

which point these cells were suitable sub culture, they were harvested

using trypsin/EDTA The MSC –like cultures demonstrated increased

prolifreration gradually and uniformly maintaining a homogeneous

fibroblastic morphology (3.6), (3.7)

3.2 Result of cryopreserved osteoblast

3.2.1.Cell viability rate and correlation with MT1, MT2, MT3 cryopreserved medium

The influence of different freezing media on the viability of osteoblast is show in Figure 2

Fig 2: Percentage of surviving cells after thawing from different

freezing media

The highest number of cell survived after thawing from freezing mediun 30% FBS and 10% DMSO, 87% The next best –performing medium was 15% FBS and 10% DMSO, 82% Also other tested media using 10% DMSO without FBS, 61% surviving cells However, the difference between difference medium was statistically significant (p<0.05)

Correlation between cell viability rate and difference freezing medium

We analyzed the cell viability of 3 groups immediately after thawing There was a correlation between the cell viability rate and MT1, MT2 groups were positive correlation Application of a straight line to this scatter resulted in y = 0,9237 X + 25.595, r = 0.437

There was a positive correlation between the cell viability rate and MT1, MT3 groups were positive correlation, correlation coefficient is r

= 0,262 The approximate straight line is y = 0.549X + 53.433

0 20 40 60 80 100