Hemovac blood after total knee arthroplasty as a source of stem cells.

BACKGROUND: With increasing life expectancy, stem cell therapy is receiving increasing attention. However, its application is restricted by ethical concerns. Hence a need exists for design of safe procedures for stem cell procurement. Here, we investigated whether hemovac blood (HVB) is an appropriate stem cell source.
METHODS: HVB concentrates (HVBCs) from 20 total knee arthroplasty (TKA) patients and bone marrow aspirate (BMA) concentrates (BMACs) from 15 patients who underwent knee cartilage repair were comparatively evaluated. A bone marrow aspiration needle was inserted into the anterior superior iliac spine. Aspiration was performed using a 50-mL syringe, including 4 mL of anticoagulant, followed by centrifugation to obtain BMACs. To obtain HVBCs, blood was aspirated from the hemovac immediately after TKA surgery. Different cell types were enumerated. Isolation of BMA and HVB mononuclear cells was performed using density gradient centrifugation. Non-hematopoietic fibroblast colonies were quantified by colony forming unit-fibroblast assay surface marker analysis of HVB, HVBC, BMA, and BMAC was performed via flow cytometry. Mesenchymal stem cells (MSCs) isolated from HVBCs and BMACs were examined for osteogenic, adipogenic, and chondrogenic differentiation potential. Gene expression analysis was performed by quantitative real-time polymerase chain reaction (qRT-PCR).
RESULTS: The number of cells from HVB and HVBC was significantly lower than from BMA and BMAC; however, the number of colonies in HVBC and BMAC did not differ significantly (P>0.05). Isolated cells from both sources had a fibroblast-like appearance, adhered to culture flasks, and formed colonies. Under different culture conditions, MSC-specific surface markers (CD29, CD44, CD90, CD105), osteogenic markers [RUNX2, osteopontin, osteocalcin, and alkaline phosphatase (ALP)] and adipogenic markers (PPARγ and C/EBPα) were expressed. Moreover, SOX9, type II collagen, and aggrecan were significantly upregulated upon chondrogenic differentiation.
CONCLUSIONS: HVB from TKA patients is a useful source of stem cells for research. 2020 Annals of Translational Medicine. All rights reserved.

Introduction

Active and continuous efforts have been undertaken in various fields to improve the quality of life as life expectancy has risen significantly over the years. Especially in the medical field, stem cell research is actively progressing in an effort to overcome intractable diseases and maintain quality health during an individual’s lifetime (1,2). For stem cell research, diverse tissues, such as umbilical cord blood, bone marrow (3), adipose tissue (4), muscle, teeth (5), cartilage, and synovium are being used as stem cell sources (6-8). Although stem cells from animal tissues can be used for research, their importance and value are lower than those from human tissues. Recently, due to ethical misconduct, reckless stem cell research has been prohibited, and strict regulatory guidelines have been reinforced by the Institutional Review Boards (IRBs) to protect patient rights. Therefore, it would not be possible to receive approval from an IRB or ethics committee if the research is found to be counterintuitive to the patients’ interests.

Total knee arthroplasty (TKA) is the gold standard for end-stage osteoarthritis, and a large number of TKA surgeries are performed each year (9,10). Blood collects inside the joint after surgery, and hence, a hemovac line is placed inside the joint post-surgery to decompress the joint, controlling pain and swelling (11-13). A hemovac drain can collect a sufficient quantity of blood to provide adequate numbers of cells. Since the blood is from both the damaged tissue and the bone cutting site, the bone marrow components are included in the hemovac blood (HVB). During a TKA surgery, cutting the surface of cancellous bones including the distal femur or proximal tibia often results in bone marrow leaking from the cut surface. Within the bone marrow aspirate (BMA), a fair amount of venous blood is also often included. Since the stromal component of bone marrow can be used to treat musculoskeletal diseases, such as nonunion fractures or cartilage defects, centrifugation and concentration of the stromal component is often performed to allow for effective clinical treatment. Similarly, HVB can also include bone marrow components; however, the amount of stromal blood is often much less than what is observed in BMAs. As such, a concentration procedure is often necessary. Hence, this study was undertaken to investigate if HVB can be manipulated in such a way to render it a useful source of stem cells for research.

We present the following article in accordance with the MDAR checklist (available at http://dx.doi.org/10.21037/atm-20-2215).

Methods

Patients

The HVB of 20 patients who underwent TKA was used for this study. The average age of the patients was 72.9 years [range, 57–86 years; standard deviation (SD), 7.56]. BMAs from 15 patients who underwent the arthroscopic cartilage repair procedure were concentrated, and the samples remaining after the procedure were used for comparative analysis. The average age of these patients was 72.1 years (range, 61–80 years; SD, 5.18). The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Institutional Review Board (UC18DESI0056) and informed consent was obtained from each patient.

Bone marrow aspiration and concentration

Bone marrow aspirate concentrate (BMAC) was obtained by inserting a bone marrow aspiration needle (SPASY™, Seoul, Korea) into the anterior superior iliac spine after sterilization under general anesthesia. Aspiration was then performed using a 50-mL syringe, including 4 mL of the anticoagulant citrate dextrose solution (Huons ACD Injection, Huons, Seongnam, Korea), after which 40 mL of BMA, including 4 mL of ACD solution, was transferred to a BMC kit (Revmed, Seongnam, Korea). Two cycles of centrifugation were performed to obtain the BMAC. The first cycle was for 6 min at 2,700 ×g, followed by a second cycle for 5 min at 2,400 ×g ().

Figure 1

The process of obtaining bone marrow aspirate concentrate (BMAC) and hemovac blood concentrate (HVBC).

HVB aspiration and concentration

Blood (40 mL), including 4 mL of ACD solution, was aspirated from the hemovac immediately after TKA surgery. The aspirated blood was transferred to the kit (BMC kit™, Revmed, Seongnam, Korea), and two cycles of centrifugation were performed to obtain the HVB concentrate (HVBC) ().

BMAC and HVBC analysis

Cell counts

The numbers of nucleated cells, lymphocytes, monocytes, red blood cells, platelets, and neutrophil granulocytes in the BMA, BMAC, HVB, and HVBC were counted using a cell counter (XE2100, Sysmex, Japan). The number of mononuclear cells (MNCs) was calculated by summing the number of lymphocytes and monocytes.

Isolation of BMA and HVB MNCs

BMAs were mixed with PBS (Gibco Invitrogen, Grand Island, NY, USA) at a 1:1 ratio. The mixture was loaded onto a histopaque layer (1.077 g/mL; Sigma chemical co., St. Louis, MO, USA). MNCs were separated by density gradient centrifugation (400 ×g, 25 min, room temperature), washed thrice with alpha minimum essential medium (αMEM; Gibco Invitrogen, Grand Island, NY, USA), filtered using a 70-µm cell strainer (Becton Dickinson, Falcon, Germany), and resuspended. The cells were incubated at 37 °C with 5% CO2 in basic medium (αMEM containing 10% FBS, 100 units/mL penicillin, 100 µg/mL streptomycin). The culture flask was washed with PBS to remove the non-adherent cells and incubated further until adherent cells reached confluence. The confluent cells were trypsinized (0.25% trypsin EDTA), divided into several culture flasks, and incubated in the basic medium. This subculture was performed for further studies, and the same procedure was repeated for HVB.

Colony forming unit-fibroblast (CFU-F) assay

A CFU-F assay was performed to quantify non-hematopoietic fibroblastic colonies. The isolated mononucleated cells were cultured in 60-mm cell culture dishes (Becton Dickinson/Falcon, Germany) for 14 days at 37 °C in a humidified atmosphere and 5% CO2. αMEM (Gibco, Life Technologies, Karlsruhe, Germany) medium supplemented with 20% FBS (Gibco), penicillin (100 U/mL; Gibco), and streptomycin (100 µg/mL; Gibco) was used for culturing.

After 14 days, adherent cells were fixed with 4% paraformaldehyde (Biosesang, Seongnam, Korea) for 5 min, washed with PBS, and stained with 1% crystal violet. The culture plate was placed on a white paper, and colonies were counted macroscopically. The number of stained cell colonies larger than 2.5 mm in diameter was counted (14).

Flow cytometry

Surface marker analysis of HVB, HVBC, BMA, and BMAC was performed using a FACSCalibur flow cytometer (BD Biosciences, Heidelberg, Germany). The harvested cells were fixed in 4% paraformaldehyde at 4 °C for 30 min and washed in flow cytometry buffer (FCB, BD Biosciences, Heidelberg, Germany). Subsequently, the cells were incubated with a blocking buffer for 30 min, followed by centrifugation to remove the blocking buffer. The cells were aliquoted at 1×106 for antibody treatment. Cells were stained with PE-labeled antibodies (Abs; CD44, CD29) and APC-labeled Abs (CD90, CD105) (BD Biosciences, San Jose, CA, USA).

Cell proliferation assay

Cell proliferation was assessed using the cell counting kit (CCK-8) assay (Dojindo, Japan). Briefly, mesenchymal stem cells (MSCs) isolated from the HVBC and BMAC were seeded at 1×105 cells/well in a 96-well plate. The assay was performed from 1 to 7 days after cell seeding. MSCs from each time point were mixed with 10 uL of CCK-8 solution/well and incubated for 2 h at 37 °C. The cellular dehydrogenase activity of the cells was then measured at 450 nm using a microplate reader. The assay was performed in triplicate.

Transwell migration assay

Migration assays were performed in Transwell plates (cat no. 3422, Corning Costar, Cambridge, MA, USA), 6.5 mm in diameter with 8 µm pore filters. P2 MSCs (5×105 cells) isolated from the HVBC and BMAC were added to the upper chamber in basal medium (αMEM without FBS). After overnight culture, 20% FBS αMEM was added to the bottom chamber. Basal medium served as a negative control. After 12 h incubation at 37 °C, with 5% CO2, the upper chamber of the filters was carefully washed with cold PBS, and cells remaining on the upper side were removed with a cotton swab. Migrated cells through the chamber, or adhered to the lower membrane, were fix in 4% paraformaldehyde. After staining with 0.1% crystal violet, the images were observed microscopically. The absorbance of migrated cells was measured at 520 nm after being dissolved in 100% methanol. Each experiment was performed in triplicate.

Multi-lineage differentiation

MSCs isolated from the HVBC and BMAC were tested for their osteogenic, adipogenic, and chondrogenic differentiation potentials. Briefly, the cells (2×104 cells/well, passage 3) were seeded in 6-well plates and treated with the osteogenic and adipogenic induction medium for 2 weeks. Culture media were changed every 3 days. For osteogenic differentiation, cultured cells were washed thrice with DPBS (Gibco Invitrogen, Grand Island, NY, USA) and fixed in 4% paraformaldehyde at 4 °C for 10 min. Mineralization of the extracellular matrix as an indicator of osteogenic differentiation was observed using Alizarin red S and alkaline phosphatase (ALP) staining (15,16).

For adipogenic differentiation, cells were washed thrice with DPBS and fixed in 4% paraformaldehyde at 4 °C for 20 min. The cells were treated with Oil Red O staining dye to visualize the lipids. For quantitative measurements, Oil Red O was eluted by dissolving in isopropanol, and the absorbance was measured at 520 nm.

For chondrogenic differentiation, the pelleted culture was used. Here, 2.5×105 cells (passage 3) were resuspended in the chondrogenic differentiation medium in 15-mL polypropylene tubes. After the cells were centrifuged at 500 ×g for 5 min to form aggregates, they were incubated at 37 °C in a humidified atmosphere of 5% CO2. The aggregates were cultivated for 21 days, and the medium was changed every 3 days. On the 21st day, the cell pellets were fixed in 10% formalin and embedded in paraffin. Sections of the cell pellets were stained with type II collagen, Alcian blue (pH 2.5), Safranin O, and toluidine blue to identify the collagen content and sulfated proteoglycans in the extracellular matrix. Hematoxylin-eosin staining was performed for morphological observation of the cells.

Adipogenic differentiation medium

This medium was comprised of the following: Dulbecco’s modified Eagle’s medium-high glucose (DMEM-HG; 11965-084, Gibco-Life Technologies, Carlsbad, CA, USA) containing 10−6 M dexamethasone, 10 µg/mL insulin, 100 µM indomethacin, and 500 µM 3-isobutyl-1-methylxanthine.

Osteogenic differentiation medium

This medium was comprised of the following: αMEM (12571-063, Gibco-Life Technologies, Carlsbad, CA, USA) containing 50 µg/mL L-ascorbic acid, 10 mM b-glycerophosphate, and 10 nM dexamethasone.

Chondrogenic differentiation medium

This medium was comprised of the following: DMEM-HG (11965-084, Gibco-Life Technologies, Carlsbad, CA, USA) containing 10−7 M dexamethasone, 10 ng/mL transforming growth factor beta 3 (TGF-β3), 100 µg/mL sodium pyruvate, 40 µg/mL proline, 25 µM ascorbic acid-2-phosphate, 100 U/mL penicillin, 100 µg/mL streptomycin, and 1% (v/v) ITS plus (5 µg/mL insulin, 5 µg/mL transferrin, 5 µg/mL selenous acid). All reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA from the MSCs of HVBC and BMAC showing osteogenic, adipogenic, and chondrogenic differentiation was extracted using the RNeasy mini kit (74104, Qiagen, Hilden, Germany). The RNA samples were reverse transcribed into cDNA using the QuantiTect reverse transcription kit (205311, Qiagen, Hilden, Germany) following the manufacturer’s instruction. All samples were analyzed using SYBR green (A6001, Promega, Madison, WI, USA) on a Promega qPCR system, and the relative expression levels were determined according to the 2−∆∆Ct method. Primer sequences used for the qRT-PCR analysis is provided in .

Table 1

List of primers used in qRT-PCR for multi-differentiation genes

Gene	Primer sequence	Product size (bp)
RUNX2 (NM001278484.2)	5'-CCG GTC TCC TTC CAG GAT-3'	122
	5'-GGG AAC TGC TGT GGC TTC-3'
ALP (NM013059.1)	5'-CCT TGA AAA ATG CCC TGA AA-3'	191
	5'-CTT GGA GAG AGC CAC AAA GG-3'
OSTCN (NM013414.1)	5'-CCT TCA TGT CCA AGC AGG A-3'	161
	5'-GGC GGT CTT CAA GCC ATA C-3'
OPN (J04765)	5'-CCT CCC GGT GAA AGT GAC-3'	71
	5'-CTG TGG CGC AAG GAG ATT-3'
CEBPA (NM176784.2)	5'-TGC GCA AGA GCC GGG ACA AG-3'	166
	5'-ACC AGG GAG CTC TCG GGC AG-3'
PPARG (NM013124)	5'-TGG AGC CTA AGT TTG AGT TTG-3'	111
	5'-ATC TTC TGG AGC ACC TTG G-3'
SOX9 (NM000.46)	5'-AGG AAG TCG GTG AAG AAC GG-3'	275
	5'-AAG TCG ATA GGG GGC TGT CT-3'
COL2A1 (J00116.1)	5'-GTT CAC GTA CAC TGC CCT GA-3'	162
	5'-TGA CCC TCA AAC TCA TGC CTC-3'
ACAN (BC150624.1)	5'-AGT CAC ACC TGA GCA GCA TC-3'	188
	5'-TCT GCG TTT GTA GGT GGT GG-3'
GAPDH (NM002046)	5'-TTG GTA TCG TGG AAG GAC TCA-3'	126
	5'-TGT CAT CAT ATT TGG CAG GTTT-3'

ALP, alkaline phosphatase; C/EBPα, CCAAT enhancer binding protein alpha; PPARγ, peroxisome proliferator-activated receptor gamma; RUNX2, runt-related transcription factor; SOX9, SRY-BOX transcription factor 9.

Statistical analysis

Statistical analysis was performed using SAS software version 9.2 (SAS Institute Inc., Cary, NC, USA). All variables were summarized using standard descriptive statistics such as mean, SD, median, and range. The Mann-Whitney test was used for comparative analyses between groups. Statistical significance is described as P<0.05.

Results

Automatic cell counts

Automatic cell counting was performed to compare the number of cells before and after concentrating BMA and HVB. The cells were increased in BMAC and HVBC ().

Table 2

Cell number comparison of hemovac and bone marrow samples

Sample	Number of samples (cases)	Sample description	Volume (mL)	WBC (×103/μL)	LYMPH (×103/μL)	MONO (×103/μL)	PLT (×103/μL)
Hemovac	20	HVB	40	7.89±2.84	1.0±0.51	0.15±0.08	31.0±13.8
		HVBC	4	28.36±19.11	4.87±3.67	0.8±0.74	125.35±97.78
Bone marrow	15	BMA	40	13.6±5.96	4.29±1.45	1.63±1.34	104.8±65.26
		BMAC	4	97.62±55.64	24.38±15.38	7.56±5.54	560.0±339.56

BMA, bone marrow aspirate; BMAC, bone marrow aspirate concentrate; HVB, hemovac blood; HVBC, hemovac blood concentrate; LYMPH, lymphocyte; MONO, monocyte; PLT, platelet; WBC, white blood cell.

Colony forming unit-fibroblast assay

CFU-F assay results demonstrate the self-renewal capacity of cells able to form new fibroblast colonies from single cells. The density and size of the colonies were found to be different according to the proliferative status of cells. A higher density and larger size of colonies indicates the higher proliferative potential of cells (14). Small fibroblastic colonies were observed on culture plates isolated from HVB and BMA at 5–7 days of culture. After 14 days, colonies were analyzed using a light microscope with crystal violet staining. The number of colonies that were >2.5 mm in size was counted (). The number of colonies were 2.30±1.87 (20 patients) in HVB, and 3.13±1.73 (15 patients) in BMA, with no statistically significant difference (P=0.365). Meanwhile, there were 14.30±9.45 (20 patients) colonies in HVBC, and 17.07±7.89 (15 patients) in BMAC, without statistically significant differences observed (P=0.187). However, the number of colonies obtained before and after concentration were significantly different in each group (P<0.05; ).

Figure 2

Colony forming unit-fibroblast (CFU-F) assay. (A) Colonies (P0) stained with 1% crystal violet on the 14th day after plating. (a) HVB (hemovac blood), (b) HVBC (hemovac blood concentrate), (c) bone marrow (d) BMA (bone marrow aspirate), (e) BMAC (bone marrow aspirate concentrate), (f) is a representative colony image. Scale bar =600 µm. (B) After 14 days of culture, colonies were counted. The number of colonies in HVB and BMA was not significantly different (P=0.365). The number of colonies in HVBC and BMAC was not significantly different (P=0.187). Hemovac n=20, bone marrow n=15; #, P>0.05. However, the number of colonies in HVB, BMA (before concentrate) and HVBC, BMAC (after concentrate) in each group showed a significant difference. *, P<0.05.

Flow cytometry

Surface expression markers in HVB, HVBC, BMA, and BMAC were confirmed by flow cytometry. CD29, CD44, CD90, and CD105, which are MSC-specific markers, were evaluated. All were highly expressed (>96%) in HVB, HVBC, BMA, and BMAC (). However, CD34 (0.18% in HVB and 0.39% in HVBC, 0.08% in BMA and 2.82% in BMAC), CD45 (0.41% in HVB and 0.49% in HVBC, 0.28% in BMA and 3.08% in BMAC), and HLA-DR (0.21% in HVB and 0.12% in HVB, 0.98% in BMA and 0.18% in BMAC) showed negative expression (<4%). shows a summary of the surface marker expression analysis of HVB, HVBC, BMA, and BMAC, which was associated with no significant variation.

Figure 3

Surface marker expression of mesenchymal stem cells (MSCs) derived from HVB, HVBC, BMA, and BMAC. (A) HVB, (B) HVBC, (C) BMA, and (D) BMAC. MSCs were positive for CD44, CD90, CD 105, and CD29 (>96%). HVB, hemovac blood; HVBC, hemovac blood concentrate; BMA, bone marrow aspirate; BMAC, bone marrow aspirate concentrate.

Isolated cells from HVB and BMA

Cells isolated from HVB and BMA were cultured and observed 3–5 days after the initial plating (a, c), and maintained in αMEM supplemented with 10% FBS and penicillin. These cells readily expanded in vitro, attached to the well, and showed a fibroblast-like morphology. The viability of the cells in each passage was greater than 98%, with no morphological changes observed (b, d).

Figure 4

Cell morphology, proliferation and migration ability. (A) Morphology of adherent cells. Cells were isolated from hemovac blood (HVB; a and b) and bone marrow aspirate (BMA; c and d). A (a and c) at 4 days, small fibroblastic mesenchymal stem cells (MSCs) were observed that were loosely arranged. A (b and d) at 11 days after initial plating, cells were confluent. Scale bar =200 µm. (B) Proliferation of cells from BMAC and HVBC incubated for 1 to 7 days and analyzed by CCK-8 assay. The proliferation rate of BMAC was higher than that of HVBC from days 4 to 7 (#, P>0.05, *, P<0.05, **, P<0.01). (C) Representative images of migrated cells for the HVBC control, HVBC, BMAC control, and BMAC. (D) Quantitative analyses of migrated cells in BMAC and HVBC (***, P<0.001, #, P<0.05).

Cell proliferation

The proliferation of MSCs isolated from the HVBC and BMAC was observed. After day 1 to 7 of culturing, the optical density (OD) of cells increased from 0.087±0.012 to 0.508±0.028 in HVBC, and 0.083±0.010 to 0.552±0.003 in BMAC (average ± SD; ). There was no significant difference between the HVBC and BMAC in OD value from days 1 to 3 (, P<0.05). Meanwhile, the OD values representing proliferation during days 4 to 7 were significantly different between the HVBC and BMAC (, P<0.05, P<0.01). The OD values were 0.383±0.022 on day 4, 0.502±0.011 on day 5, 0.519±0.009 on day 6 in HVBC, and 0.451±0.016 on day 4, 0.540±0.016 on day 5, and 0.553±0.004 on day 6 in BMAC. The pattern of increasing OD values for cell numbers between the HVBC and BMAC was similar.

Cell migration

Next, the migration ability of MSCs isolated from the HVBC and BMAC was observed using crystal violet staining. The cells from the HVBC and BMAC were morphologically similar (). The measurement of the OD value of stained cells represents the number of migrated cells. The OD value of 20% FBS αMEM was 1.182±0.024, which was approximately 2.2-fold higher than the control (αMEM without FBS; 0.538±0.045) in HVBC. Meanwhile, the OD value of 20% FBS αMEM was 1.18±0.019, and approximately 2.3-fold higher than the control (αMEM without FBS; 0.515±0.019) in BMAC. There was no significant difference between the HVBC and BMAC in OD values (, P<0.05).

Multi-lineage differentiation (chondrogenic, osteogenic, and adipogenic)

Adipogenic differentiation was confirmed by Oil Red O staining to identify intracellular lipids. Intracellular lipid droplets in isolated cells from HVB and BMA were observed. Lipid droplets were not found in the undifferentiated condition (). Absorbance values were 0.311±0.011 nm for HVBC, and 0.371±0.051 nm for BMAC in the undifferentiated condition, and 0.945±0.051 nm for HVBC, and 0.860±0.051 nm for BMAC in the differentiated condition (). Absorbance values in the undifferentiated and differentiated conditions between HVBC and BMAC were not significantly different (P>0.05). However, absorbance between the undifferentiated and differentiated conditions for HVBC and BMAC were significant (P<0.05; ).

Figure 5

Adipogenic differentiation of mesenchymal stem cells (MSCs) derived from HVBC and BMAC on the 14th day of culture. (A) The accumulation of intracellular lipid drops was observed by Oil Red O staining, indicating adipogenic differentiation. Scale bar =100 µm. (B) Absorbance results of HVBC (average 0.945±0.051 nm) and BMAC (average 0.86±0.051 nm) in the differentiation condition were not significantly different (P=0.108). n=3, #, P>0.05, ***, P<0.001. HVBC, hemovac blood concentrate; BMAC, bone marrow aspirate concentrate.

Osteogenic differentiation was identified by Alizarin red S (calcification of the extracellular matrix) and ALP staining (). Calcification during osteogenic differentiation appears as large crystal clusters among cells. Crystal clusters (calcification in the extracellular matrix) were stained red with Alizarin red S staining (). Absorbance values were 0.044±0.001 nm for HVBC and 0.042±0.001 nm for BMAC in the undifferentiated condition and 0.419±0.012 nm for HVBC and 0.516±0.013 nm for BMAC in the differentiated condition. Absorbance values between HVBC and BMAC in the differentiated condition were significantly different after Alizarin red staining (P<0.001) with a higher value in BMAC (). The absorbance value of the undifferentiated and differentiated conditions between HVBC and BMAC in the ALP assay did not show any significant difference with values of 47.025±0.837 U/mL in HVBC, and 48.548±3.195 U/mL in BMAC (P>0.05; ).

Figure 6

Osteogenic differentiation of mesenchymal stem cells (MSCs) derived from HVBC and BMAC on the 14th day of culture. (A) Alizarin red S was used to observe calcium deposition. The blue color represents alkaline phosphatase. Scale bar =200 µm. (B) Absorbance results of Alizarin red S increased after differentiation as compared to that before differentiation. Alkaline phosphatase activity increased after differentiation as compared to that before differentiation, and the results were significantly different (Alizarin red S; ***, P<0.001, alkaline phosphatase: *, P<0.05). HVBC, hemovac blood concentrate; BMA, bone marrow aspirate; BMAC, bone marrow aspirate concentrate.

To evaluate chondrogenic differentiation, hematoxylin-eosin staining was performed to observe the morphology of cells, and Alcian blue pH (2.5) and toluidine blue staining was performed to observe the pericellular proteoglycan and type II collagen deposition (). Many round cells with surrounding lacuna were observed after hematoxylin-eosin staining (black arrow, ). The rich extracellular matrix surrounding the cells was observed in the chondrogenic differentiation group of both HVBC and BMAC (). The pellets in the differentiated condition showed strong toluidine blue () and Alcian blue pH (2.5) staining (indicative of cartilage matrix; # ). Safranin O staining and the morphology of the cells suggested that a cartilaginous matrix had been synthesized. The highly stained area after toluidine blue and Alcian blue pH (2.5) staining corresponded to the safranin O-stained areas in the chondrogenic differentiation condition (). Type II collagen expression inn HVBC and BMAC was confirmed via immunohistochemical staining (). Type II collagen is the main collagen component of the extracellular matrix and comprises the cartilage-specific matrix (17,18). Type I collagen was not detected in any of the groups ().

Figure 7

Chondrogenic differentiation of mesenchymal stem cells (MSCs) derived from HVBC and BMAC on the 21st day of culture. MSCs were cultured in the form of a pellet (2.5×105 cells per pellet). Hematoxylin-eosin (H&E) staining of the pellets (A-D); Safranin O (E-H); Alcian blue (pH 2.5) (I-L); Toluidine blue (M-P); immunohistochemical staining for detection of type II collagen Q-T) and type I collagen (U-X). elongated cells were seen in the non-differentiation group (black arrowhead, A,C) and chondrocyte-like cells with lacuna were observed in the chondrogenic differentiation group (black arrow, B,D). Strong staining of safranin O (F,H), Alcian blue (pH 2.5) (blue stain # of J,L), toluidine blue (purple stain ** of N,P) and type II collagen (R,T) demonstrated the formation of hyaline cartilage-like tissue in the pellets of the chondrogenic differentiation group. Scale bar =100 µm. HVBC, hemovac blood concentrate; BMA, bone marrow aspirate; BMAC, bone marrow aspirate concentrate.

Gene expression

Total RNA was extracted from MSCs in HVBC and BMAC on the 14th day of culture after osteogenic and adipogenic differentiation, and on the 21st day of chondrogenic differentiation. Expression of the genes for runt-related transcription factor 2 (RUNX2), osteopontin (OPN), osteocalcin (OSTCN), and ALP, were analyzed as osteogenic differentiation markers. RUNX2, OPN and ALP expression levels were higher in BMAC than in HVBC. However, OSTCN expression levels in HVBC and BMAC were not significantly different (; P<0.05, P>0.05). Further, the adipogenic differentiation markers PPARG and CEBPA were observed. The expression of PPARG and CEBPA genes after adipogenic differentiation was upregulated; however, the difference between HVBC and BMAC was not significant (, P>0.05). Additionally, the genes for type II collagen (COLA1), aggrecan (ACAN), and SOX9 were investigated as chondrogenic differentiation-specific markers (). The expression of all three was significantly upregulated in the chondrogenic differentiation group; however, the difference between HVBC and BMAC was not statistically significant (, P>0.05).

Figure 8

Comparison of multi-differentiation gene expression level between HVBC and BMAC. (A) Expression of osteogenic differentiation genes (RUNX2, osteocalcin, osteopontin and alkaline phosphatase) at 14 days of osteogenic differentiation culture. RUNX2, osteopontin, and alkaline phosphatase gene expression in BMAC was higher than that of HVBC (*, P<0.05). Osteocalcin expression was not significantly different between the groups. (B) Expression of adipogenic differentiation marker genes (C/EBPα and PPARγ). No statistically significant difference was observed between HVBC and BMAC. (C) Chondrogenic differentiation marker gene expression (SOX9, aggrecan, type II collagen). No statistically significant difference was observed between HVBC and BMAC (#, P>0.05). HVBC, hemovac blood concentrate; BMA, bone marrow aspirate; BMAC, bone marrow aspirate concentrate.

Discussion

Since stem cell research is an actively progressing field, finding an adequate cell source is critical. Bone (19), bone marrow (20), synovium (21,22), adipose tissue (23), cord blood (24), are well known sources (25-27). Additionally, the nasal septum, muscle, and cartilage serve as stem cell sources for musculoskeletal regeneration (28-31).

Bone marrow mesenchymal stem cells (BMSCs) have served as the primary source of stem cells for many years. However, harvesting BMSCs is a painful procedure and they exhibit signs of senescence at an early stage of expansion compared with MSCs derived from other sources (32,33). Nevertheless, BMSCs require a relatively short culture period (34,35), and many clinical studies using this source are actively being conducted. Alternatively, adipose tissue derived MSCs can be readily isolated (35) with morphological and phenotypical characteristics similar to BMSCs, which are stable during the culture period (36). Moreover, although umbilical cord blood derived MSCs (UCB-MSCs) have an associated long cultivation period, they exhibit high proliferation capacity (37,38). Meanwhile, peripheral blood derived MSCs are easily obtained, which is the reason for their application in many animal studies (38-45), however, they are present at low levels in mononuclear cells (38).

Compared to the other sources of stem cells, HVB can be readily obtained after various bone surgeries, including TKA, without the need for special procedures that can cause additional pain and be met with ethical issues. Although the amount of HVB may vary between patients, a sufficient number of MSCs can be obtained through concentration and cultivation of the cells. Nevertheless, the hemovac must be treated carefully and aseptically following TKA. Should the HVB become contaminated, the hemovac must be removed immediately from the patient to avoid the complications associated with TKA infection. Hence, to be used as a regular source of stem cells, an advanced care protocol for hemovac is required.

TKA is a widely performed surgery and considered to be the gold standard for treatment of late-stage osteoarthritis (9,10). During this surgical procedure, the degenerated bone and cartilage are cut, thereby releasing a large amount of bone marrow components from the bone cutting surface. After the operation, the hemovac line can be inserted intra-articularly to decompress the knee joint thereby preventing hematoma formation. The blood collected in the hemovac contains bone marrow components, which can be used for stem cell research. Most importantly, HVB can be obtained without causing additional pain or harm to the patient. Through the centrifugation of HVB, a sufficient number of cells that have multi-lineage differentiation capacity as stem cells can be obtained. Although the number of cells from HVB and HVBC was very low compared to that from BMA and BMAC, the number of colonies from HVBC and BMAC was not significantly different (#, P>0.05). These cells are actual participants in tissue regeneration.

The cells derived from HVB adhered to the culture flask with fibroblast-like morphology, showed multi-lineage differentiation, and expressed stem cell markers (46). CD marker analysis of HVB and HVBC showed over 96% expression of positive markers (CD29, CD44, CD90, CD105) of MSCs. These results were very similar to those obtained with BMA and BMAC and showed no significant differences.

The multi-differentiation potentials of HVB were evaluated through the culture of cells derived from HVB in osteogenic, adipogenic, and chondrogenic induction medium (46). Alizarin red S (calcium deposit, mineralization) and ALP activity were used for osteogenic differentiation evaluation; BMA showed a higher level of Alizarin red staining, indicating calcium deposit and mineralization. However, the difference in ALP activity between HVB and BMA was not significant. Higher gene expression in BMA was observed for RUNX2, ALP, and osteopontin, but not for osteocalcin.

Peroxisome proliferator-activated receptor-γ (PPARγ) plays an important role in adipogenic differentiation (47). Lipid drop formation was observed using Oil Red O staining, and PPARγ and C/EBPα, as adipocyte differentiation markers, were expressed in HVB and HVBC. The Alcian blue (pH 2.5), safranin O, toluidine blue, and type II collagen staining of cells in HVB and HVBC showed chondrogenic differentiation in the form of characteristic matrix synthesis and the formation of cells with surrounding lacuna. Additionally, specific chondrogenesis markers (1) of gene expression, such as type II collagen, SOX9, and aggrecan, were upregulated after 21 days of culture in chondrogenic conditions. The expression of SOX9, aggrecan, and type II collagen did not show any statistically significant difference between HVB and HVBC.

The stem cells from HVB showed characteristic multi-potentiality, and a sufficient number of cells could be obtained through centrifugation without any harm or pain to the patients. Through this study, we comprehensively demonstrated that the stem cells from HVB could be potentially used in stem cell research involving musculoskeletal regeneration, including that of cartilage, bone, and fat. Moreover, these stem cells can also be explored for their clinical application potential.

After undergoing a TKA operation, patients return to their beds from the surgical theater with a hemovac placed in the knee joint. If the hemovac becomes full of blood, the contents are discarded and new blood begins to fill the hemovac. These first two hemovacs serve as potential sources of stem cells, as after this, the majority of the content will consist of venous blood. The hemovac can hold up to 400 mL of blood, and the kit that was used in this study processes 40 mL. Hence, theoretically, 800 mL can be collected from the first and second hemovac, which can be subsequently concentrated using 20 kits. Each kit can produce 4 mL of HVBC with counts obtained as follows: white blood cells, 28.36±19.11×103/µL; lymphocytes, 4.87±3.67×103/µL; monocytes, 0.8±0.74×103/µL; platelets, 125.35±97.78×103/µL (). It is, therefore, possible to ultimately collect 20 times more than each of these cell counts from a single patient with this method.

HVBC can produce modified autologous stem cells that can be stored for future clinical application. Moreover, if necessary, cells can be cultured to produce enough cells for clinical applications. Therefore, this method might prove to be practical and efficient for clinical trials. For example, if patients suffer from nonunion fractures, osteonecrosis of the femoral head, or osteoarthritis of the knee joint, we can use these banked stem cells as therapeutic options. However, for this kind of clinical application, more laboratory studies are required and clinical trials must be performed.

Recently, studies have been reported on the injection of BMAC into knee joints (48-53). However, bone marrow aspiration must be performed in the operation theater under aseptic conditions with adequate anesthesia according to patient’s condition. Further, this procedure is also painful. If HVBC from unilateral TKA can be stored, the cells from this source can be injected into the contralateral knee joints to improve the patient’s arthritis-related symptoms.

In this study, we also investigated the optimal time for HVB harvesting. Theoretically, that obtained immediately after surgery is considered of higher quality than that collected later. However, more stem cells were observed in the second HVB aspiration than in the first. There is a possibility that wound irrigation with normal saline prior to completing the operation might dilute the cells from the bone marrow.

To the best of our knowledge, there has been no previous report regarding MSCs derived from HVB. This study demonstrated the morphology, proliferative potential, surface markers, and multi-differentiation ability of the cells obtained from this source, and every aspect was found to be comparable to that of bone marrow-derived stem cells. In conclusion, HVB obtained after total knee replacement can be used as a source of stem cell research or autologous stem cell therapy without the need for invasive and painful bone marrow aspiration procedures.

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