Induced-Coagulated Plasma-Fibrin Gels as a Biological Scaffold for Cell Attachment and Proliferation of Umbilical Cord-Derived Mesenchymal Stem Cells (UC-MSC)

Fibrin gels are an ideal natural biological scaffold for tissue engineering because they are biocompatible, biodegradable, and have many biological surface markers. However, most research on fi brin gels used commercial fi brin kits that could be costly and limited in some areas. In this study, fi brin gels were made by inducing blood coagulation by adding a common diagnostic kit to assess the time for blood to clot, called activated partial thromboplastin time (aPTT). This induced coagulated plasma (iCoplas)-fi brin gels was evaluated for its ability to enhance biological activity of umbilical cord-derived mesenchymal stem cell (UC-MSC), which were cell attachment and proliferation. Fibrinogen concentration had infl uence on cell attachment, where only 50% of the cells could attach to 77 mg/dl fi brinogen gels whereas 93% cells adhered to 154 mg/dl fi brin gels. There were no signifi cant differences in cell proliferation on polysterene culture dish and fi brin gels (p>0.05). These results showed that iCoplas-fi brin gels could be used as a fi brin-based scaffold, yielding no signifi cant difference than polysterene-tissue culture dish cultures in cell attachment and cell proliferation on 154 mg/dl fi brinogen concentration.


Introduction
Bone defects could be caused by disease, degenerative process, or trauma.Treatment using cells or materials from the patient and implant it to the damaged tissue are called autologous treatment.Autologous bone grafts are often used to treat bone defects.Although this method can produce good results, it has drawbacks such as second site morbidity, pain, increased risk of surgical infection, and limited graft supply.Bone substitution method is an alternative method that can be used to treat bone defects.
Physical characteristic of bone substitutes; such as porosity, pore size, and surface availability, should be considered so that the substitute could imitate the condition and characteristic of the bone.These characteristic is essential for cell attachment, proliferation, and differentiation of the cell to generate new bone tissues (Castelas et al., 2006).
Tissue engineering is an interdisciplinary fi eld that applies the principle of engineering and life science, in the purpose of developing a biological substitute that can restore, maintain, or improve tissue function or a whole organ.Tissue engineering consist into three components; cells, scaffold, and signal molecules (Ohba et al., 2009).Cells that are usually used in tissue engineering are stem cells.Stem cells are undifferentiated cells that can be differentiated into many other cells.Stem cells also have the ability to self renew (Meirelles et al., 2009) making it as an advantage for tissue engineering.
Mesenchymal stem cells are adult stem cells, commonly found in the bone marrow and umbilical cord (Docheva et al., 2008).Adult mesenchymal stem cells are suitable for clinical use as they could attach to plastic, proliferate, and differentiate well in vitro, and have suitable properties for implantation by having low immunogenic and high immunosupressive properties (Fosset and Khan, 2011).Umbilical cord-derived mesenchymal stem cells are much harder to isolate but had a superior proliferation potential and more suppressive effects on peripheral blood mononuclear cell proliferation than bone marrow-derived mesenchymal stem cells (Arufe et al., 2011).
Scaffolds are tailored made matrix to mimic natural environment for the cell, giving it an optimal condition to attach, proliferate, and differentiate (Baker and Chen, 2012).Fibrin, a natural occurring biodegradable matrix, is reported to be biocompatible, biodegradable, and has many biological surface marker making it an ideal natural biological scaffold (Shaikh et al., 2008).Several researches on tissue engineering have explored the potency of commercial fi brin kit to make fi brin-based scaffolds.Although benefi ts in effi ciency, using commercial fi brin kit could be costly and are not always available in some places.Activated Partial Thromboplastin Time (aPTT) Kit is a coagulation diagnostic kit to assess the prothrombin time or the time for blood to clot in clinical setting (Korte et al., 2000).Using aPTT kit to fabricate induced coagulated plasma (iCoplas)-fibrin gel scaffolds, opens the possibility to use the patient's own blood as autologous scaffolds which could decrease the potential risk of body reaction (Ye et al., 2000).In this study, we observe cell attachment and proliferation characteristic of umbilical cord-derived mesenchymal stem cells in iCoplas-fibrin gels.

Ethical Acceptance from the Etchical Committee Board
This study had been approved by the Institutional Review Board at the Faculty of Medicine, Universitas Gadjah Mada (UGM), Yogyakarta for collection and study with human umbilical cord tissue.

Sample Criteria
Umbilical cord was taken from aterm babies (32 until 38 weeks old) born in hospitals or clinics that cooperate with this research.Inform consent was informed and signed by the parents.Samples are stored in a sterile 50 ml centrifuge tube containing Phosphate Buffer Saline (PBS) (Biobasic, Toronto, Canada) supplied with penicillin (Gibco, 15140, Invitrogen Corporation, NJ, USA) and fungizone (Gibco, 15140, Invitrogen Corporation, NJ, USA).Samples are processed immediately or stored in 4 o C for a maximum 24 hours.

P l a s m a C o l l e c t i o n a n d F i b r i n o g e n Concentration Measurement
Whole blood for plasma source were obtained from healthy donor, collected in citrate vacutainer vacutainer (BD Vacutainer, NJ, USA) and processed by centrifugation at 2000 rpm for 10 minutes at room temperature.Fibrinogen concentration in the collected plasma citrate was measured by using turbidimetry method in Clinical Pathology Laboratory, Sardjito Hospital, Yogyakarta.

Icoplas-fi brin Gel Fabrication
Plasma citrate was diluted with double distilled water to obtain 154 mg/dl and 77 mg/dl fi brinogen concentration.Fibrin gels were fabricated by adding 150 μl of plasma and 100 μl TriniCLOT aPTT HS (Tcoag, Ireland) into a 24-wells plate.To homogenize the suspension, the plate was agitated gently then incubated in 37 o C for 5 minutes then the solution was added by 150μl of warm 0.1 M CaCl 2 and incubated at room temperature for 30 minutes.Fabricated fi brin gels were rinsed with phosphate buffered saline and sterilized by ultraviolet (UV) radiation for 1 hour.

Isolation from umbilical cord by Explant Method
Cells was isolated using the explant method, according to Koliakos et al. (2011) without any addition in enzymatic reaction.Umbilical cord samples were cut into 1 cm 3 pieces and washed with sterilize PBS twice.Each explant pieces were cut again into 3 mm 3 explants and washed with Dulbecco's Modifi ed Eagle Media low glucose (DMEM-LG) (Gibco, 25200, Invitrogen Corporation, Canada).Explants were placed into 60 mm culture dish and was incubated in room temperature for 3 minuets.The cells were cultured with DMEM-LG medium supplemented with Fetal Bovine Serum (FBS) 10% (v/v) (FBS Heat Inactivated, Caissonlabs, USA), penisilin streptomisin 1% (v/v) , dan fungizone 0.5 %.Cell culture was incubated in 37 o C, 5% CO 2 with 90% humidity.Culture medium was changed every two days and the cells were not used past passage 4. Upon reaching 100% confulency, cultured cells were trypsinize and subcultured.

Detection of CD45 expression by a.
immunofl owcytometry Approximately 10 5 umbilical cordderived cells were centrifuged at 2000 rpm for 5 minutes.Pellet cells then were added with 2.5 μl anti-CD45 (BD Bioscience, San Jose, CA 95131, USA) and vortex for 2 minutes.Cells were then incubated in dark at room temperature.Pellet cells were then washed with 1 ml of wahing buffer and centrifugated at 2 000 rpm for 5 minutes.Pellets cells were then resuspended with 100 ml Facsfl ow.Cell suspension then transfered to a fl owcytometry tube and analyzed using FacsCalibur.

Detection of STRO-1 expression by b. immunocytochemistry (ICC)
Cells with a density of 5x10 4 cells/ ml was seeded on a cover slip in a 35mm TCD and incubated until 70% confl uence.Cells on the cover slip were fi xed with cold methanol and 0.3% H 2 O 2 .Cover slip were then washed with PBS and added with background snipper and incubated in room temperature for 10 minutes.The STRO-1 primary antibody were added to the cover slip and incubated for 10 minutes.Cover slip were washed again with PBS and added with 100 μl biotinylated universal secondary antibody (Novostain Universal Detection Kit NCL-RTU, Novocastra Lab LTD, UK) and incubated for 10 minutes.Cover slip were washed again with PBS and added with Betazoid DAB chromogen and incubated for 10 minutes.Cover slip were washed with aquadest and added 2 μl of counterstain MayerHaematoxylin (Dako, Denmark) and incubated for 30 seconds.Cover slip were then washed with aquadest and dip in xylol then alcohol before incubating it in room temperature to dry.The dried cover slip is then mounted on the object glass and observed under the microscope.Cells with STRO-1+ will be colored dark brown as for negative cells will be blue.

Cell attachment assay on PS-TCD and fi brin gels
Approximately 10 4 cells are cultured in a 24 well plate with or without fibrin gels.Cells were supplemented with growth medium and incubated in 37 o C for 1 and 3 hours.Cells were then trypsinized and cell count was done by using hematocytometer.

Cell proliferation characteristic using MTT assay
Approximately 10 4 cells are cultured in a 24 well plate with or without fi brin gels.Cells were then incubated in 37 o C for 1, 3, 5, and 7 days.Cell proliferation was investigated using the MTT assay (refer to Song et al., 2013).Medium was changed in every 2 days.

Osteogenic diferentiation of UC-MSC
Approximately 3x10 4 cells are cultured in a 24 well plate.Cells were then supplemented with growth medium and incubated in 37 o C. On the second day, growth medium was changed with osteogenic medium which contained DMEM medium, 10% FBS, 150 μg/ ml ascorbic acid, 10 mM β-glycerophosphate and10 nM dexamethasone (Silla-Asna et al., 2007).

Statistical analysis
All results represent replicates (n=3).Statistical analysis was performed using oneway Analysis of Variance (ANOVA) followed by Fisher's paired Least Signifi cance Difference (PLSD) and analyze with Student t-test; a p value <0.05 was considered to indicate statistical signifi cance.

Umbilical cord-derived cells, obtained by explant method, express STRO-1 marker and did not express the hematopoietic marker, CD45
Fibroblast-shaped cells were isolated from the umbilical cord using the explant method.Cell surface markers were investigated to characterize the isolated cells.STRO-1, a proven specifi c mesenchymal stem cell marker (Bobis et al., 2006), was expressed in cultured cells using the immunocytochemistry staining as shown by stained brown cells (Figure 1B).CD45, a well known hematopoietic marker, was also investigated using immunofl owcytometry (Docheva et al., 2008).There were 98.24% cells that did not express the CD45 marker.
Mesenchymal stem cells are a well investigated stem cell due to their unique characteristic.However, there are no defi nite specifi c marker for the identifi cation of MSC (Docheva et al., 2008).To avoid uncertainty, in 2006, the International Society for Cellular Therapy had proposed minimal criteria to define MSC.First, MSC must have the ability to attach to plastic in standard culture conditions.Second, MSC must express CD73, CD90, and CD105 and do not express CD11b, CD14, CD19, CD34, CD45, and HLA-DR surface markers.Third, MSC must be able to differentiate into osteoblast, adipocyte, and chondrocyte in vitro (Dominici et al., 2006).Fibroblast-like cells were obtained from umbilical cord explant.The cells adhered to plastic and were expand until 5 passages.Immunofl owcytometry using CD45 antibody showed 98.24% of the cells did not express the CD45 surface marker.
The ability to differentiate into osteoblast was also evaluated by mineralization Alizarine Red Staining.Cells culture showed change in morphology and increase mineralization after 14 days of incubation in osteogenic medium (Figure 1D).Mineralization was an indicator for osteoblast maturation and is considered as a osteogenic differentiation test (Silas-asna et al., 2007).These results indicate that the umbilical cord-derived cells, isolated by explant culture method, could possibly be MSC.

iCoplas-fi brin gel could be degraded after 4 weeks in basal medium
Icoplas-fibrin gels were successfully fabricated using the aPTT kit (Figure 2A).Fibrinogen concentration from plasma citrate from a single donor was evaluated.Citrate plasma from the donor had 154 mg/dl of

A B C D
fi brinogen concentration and is used for the main concentration to fabricate the fibrin gels.In 2008, Ho and coworkers, state that fi brinogen concentration of fi brin gels could infl uence cell proliferation.To evaluate this matter, plasma citrate was diluted with aquabidest to 77 mg/dl concentration.Fibrin gel was formed after incubation of citrate plasma, aPTT HS, and CaCl2 0.1M.Incubation time for clot formation were 7 minutes and 40 seconds and 12 minutes and 35 seconds for fi brin gel with a concentration of 154 mg/dl and 77 mg/ dl, respectively.Fibrin gel degradation assay was conducted by incubating fi brin gels in growth medium and was observed visually under the microscope.The 77 mg/dl fi brin gel showed fi brin degradation after 10 to 14 days of incubation in basal medium as for the 154 mg/dl fi brin gel showed no signs of degradation after 4 weeks of incubation in basal medium (Figure 2).Higher fibrinogen concentration took longer time to degrade.Fibrin gel degradation is a natural process, which conclude restriction of cross-linking bonds and monomer release.In the body, fi brin gels or fi brin clots are broken down by plasmin, an enzyme produced by the liver, resulting in the release of fi brin degradation products (FDP) (Cesarman-Maus and Hajjar, 2005;Ho et al., 2006).In vitro cell culture fi brin degradation could be cause by unstable or low forming cross-linking bonds.Low  sequence, R-G-D (arginin-glycine-aspartate acid) which could be recognize by specifi c cell receptors called integrin (Lee et al., 2004;Ogura et al., 2004).This pairing of site specifi c motif and integrin will mediate cell attachment and the formation of focal adhesion (Frantz et al., 2010).Lower cell attachment could be cause by lower concentration of fi brinogen, limiting the availability of site-specific sequences, which are signifi cant for cell attachment.

Cell proliferation did not give signifi cant difference in PS-TCD and fibrin gel scaffolds
Cell proliferation was measured day 1, 3, 5, and 7 in a 24-well plate.Starting density was 5 x10 3 cell/cm 2 .Proliferation characteristic in PS-TCD and fibrin gels gave no signifi cant differences.All cultures showed similar patterns in cell proliferation.Day 1, there were a decrease of viable cells in all cultures.In fi brin gels viable cell number decrease until 80.4% and 43.16% in 154 mg/ dl and 77 mg/dl fi brin gels, respectively.Huang and co-workers (2010), proposed that trypsin induce proteome alteration during cell subculture.Furthermore, they found out that 36 proteins were expressed differently on trypsinized cells, where proteins involve in cell metabolism, growth regulation, mitochondrial electron transportation, and cell adhesion are down-regulated and proteins involve in cell apoptosis are upregulated.Cells that were transported using the trypsinize method would undergo apoptosis mechanism resulting in a decrease of viable cells.
An increase in cell proliferation was observed from day 3 until day 7 for PS-TCD and fi brin gel 154 mg/dl cultures.Fibrin gel 77 mg/dl cultures had increase in viable cell until day 5, and decreases on day 7 (Figure 3).
On day 7, cells cultured on PS-TCD showed more viable cells than fibrin gel cultures.Variation of the ECM properties such as topography, elasticity, and stiffness could infl uence cell behavior (Tam et al., 2012).concentration of fi brinogen could form fewer cross-linking bonds resulting in a shorter time of degradation.

Fibrinogen concentration influence cell attachment
Cell attachment was measured by cell count after 1 and 3 hours of cell seeding into the fi brin gel with 154 mg/dl and 77 mg/dl fi brinogen concentration.Polysterene tissue culture dish (PS-TCD) was used as a control as mesenchymal stem cell would attached to plastic surface (Ogura et al., 2004).After 1 hour of seeding, 50%, 43%, and 37.5% cells were attached to PS-TCD, fi brin gel 154 mg/ dl, and fibrin gel 77 mg/dl, respectively.Cells attached after 3 hours of incubation statistically, showed no signifi cant difference in PS-TCD, 87.5%, and fi brin gel 154 mg/dl, 93%.As for fi brin gel 77 mg/dl showed much less cell attachment with only 50% cells were attached to the scaffold.
Mesenchymal stem cells adhered to plastic and to fi brin gels.Morphologically there are differences in the PS-TCD and fi brin gel cultures.In PS-TCD, the cells form a more fl attened shaped and in the fi brin gels the cells forms a thinner spindle liked shaped.Cell attachment onto extracellular matrix (ECM) will form focal adhesions bridging the ECM and the cytoskeleton of the cell (Tam et al., 2012).Focal adhesion is an integrated center for cell signaling, mechanosensing, and force transduction to mediate cell attachment, spreading, and motility in response to ECM (Fraley et al., 2010).They consist of multiprotein complexes (e.g.talin, vinculin, and paxillin), and transduce the mechanical response to dynamic remodeling of actin cytoseleton.Variation in composition and physical characteristic of the matrix could infl uence cell shape and function due to this mechanical feedback from the ECM to cell behavior (Tam et al., 2012;Trappmann et al., 2012).
Cell attachment assay showed fi brinogen concentration in fi brin gels could infl uence cell attachment.Fibrin possess a site-specifi c Evans and co-workers (2009) conclude that cell growth, proliferation and differentiation were all increased as a function on ECM stiffness.PS-TCD cultures have higher stiffness than fi brin gel cultures, giving it an advantage in cell growth and proliferation.However, there are more parameters that need to be considered for tissue cell culture.
Dimensionally, fibrin gels forms a 3-dimensional (3D) structure giving it dissimilarity characteristics than the 2-dimensional (2D) PS-TCD.Baker and Chen (2012) proposed that the difference in dimensional structure of 3D and 2D could effect on soluble gradients, polarity, matrix properties, adhesion molecules, and matrix stiffness which all then could infl uence on cell attachment, growth, proliferation, and differentiation.

Conclusion
STRO-1 + /CD45 -mesenchymal stem cell was isolated from the umbilical cord using the explant method.These fi broblast-shaped cells could attached to plastic and differentiate into osteoblast cells.Icoplas-fi brin gels could be fabricated using the aPTT kit with citrate plasma with fi brin concentration of 154mg/ dl and 77 mg/dl.Cell attachment was infl uenced by fi brinogen concentration as for higher fi brinogen concentration (154 mg/dl) gave no signifi cant difference then PS-TCD culture.ECM plays a role in cell proliferation, PS-TCD culture with higher stiffness showed higher proliferation although did not gave signifi cant difference.These results showed that iCoplas-fi brin gels could be used as a fi brin based scaffold yielding no signifi cant difference than PS-TCD cultures in cell attachment and cell proliferation on 154 mg/ dl fi brinogen concentration.It is exciting to see rather or not iCoplas-fi brin gels share differentiation potential than PS-TCD.Further research on this matter could lead to the possibility of develop an autologous treatment for bone engineering.

Figure 1 .
Figure 1.Identifi cation of umbilical derived-cells.(A) Immunofl owcytometry using CD45 antibody showed 98.24% cells are negative; (B) Immunocytochemistry using STRO-1 antibody gave STRO-1+ cells a dark brown colour pointed by white arrows.(C) Colony forming unit fi boblast (CFU-F) forms in 3 day cultures pointed by dotted arrows; (D) Alizarine staining culture in osteogenic medium showed mineralization by formation of red nodules pointed by black arrows.Bars : 50 μm.

Figure 2 .
Figure 2 .Fibrin gel made by using aPTT Kit.(A) Fibrin gels forms by adding plasma citrate, aPTT HS, and CaCl2.(B) Fibrin gel degradation, with 77 mg/dl fi brinogen concentration, visualized using inverted microscope after 10 to 14 days of incubation with growth medium.Bars : 50 mm.

Figure 3 .
Figure 3. Mesenchymal stem cell characteristic on fi brin gels.(A) Cell attachment assay, cell attachment on fi brin gels are infl uenced by fi brinogen concentration; and (B) Proliferation assay showed no signifi cant difference in PS and fi brin gel cultures.