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OPEN

Characteristics of autologous
protein solution and leucocytepoor platelet-rich plasma for thetreatment of osteoarthritis of theknee
Shiho Wasai1,2, Masato Sato1,2 ✉, Miki Maehara1,2, Eriko Toyoda1,2, Ryoka Uchiyama1,2,
Takumi Takahashi1,2, Eri Okada1,2, Yoshiko Iwasaki1,2, Satoko Suzuki1,2 &Masahiko Watanabe1,2Recently, platelet-rich plasma (PRP) has received attention as a treatment for patients withosteoarthritis of the knee (OAK), a chronic degenerative disease, to bridge the gap between
conservative and surgical treatments. Here, we investigated the differences in the humoral factors
present in two types of PRP purified using the Autologous Protein Solution (APS) kit (group Z;
leucocyte-rich PRP) or the Cellaid Serum Collection Set P type (group J; leucocyte-poor [LP]-PRP).
Differences in humoral factors between healthy subjects (n = 10) and OAK patients (n = 12; group
Z = 6, group J = 6), and the relationship between humoral factors and clinical outcome scores were
investigated. Both anti-inflammatory and inflammatory cytokines were highly enriched in APS. The
concentrations of tumour necrosis factor (TNF)-α, platelet-derived growth factor, fibroblast growth
factor, soluble TNF-receptor 2, soluble Fas and transforming growth factor-β1 were higher in group Z,
while the total amounts were higher in group J. The concentration of interleukin-1 receptor antagonist
was positively correlated with the magnitude of change in the clinical outcome score and may
contribute to improving knee-joint function. This is the first description of the humoral factors in APS
and LP-PRP prepared from healthy subjects or OAK patients of Asian descent.
Osteoarthritis of the knee (OAK) is a chronic degenerative disease that progresses slowly with age. There are
estimated to be 25.3 million patients with OAK in Japan. The number of symptomatic patients is estimated at 8
million and is expected to continue to increase with Japan’s hyper-aging population1.
Current methods of treatment for patients with OAK include patient education, exercise, oral treatments(i.e. non-steroidal anti-inflammatory drugs [NSAIDs], acetaminophen, tramadol), conservative therapy byintra-articular injection of hyaluronic acid or steroids or surgical treatments, including arthroscopic debridementof the knee joint, osteotomies around the knee such as high tibial osteotomy, unicompartmental knee arthroplasty(UKA) and total knee arthroplasty (TKA)2,3.
In recent years, cell therapies and platelet-rich plasma (PRP) have been investigated as treatments to bridge thegap between conservative and surgical treatments. Intra-articular injection of PRP for the treatment of OAK has
been reported to relieve pain, suppress inflammation and regenerate cartilage4–7. A variety of commercial kits for
preparing PRP are available, and in Japan, the use of PRP therapy is gaining popularity. The blood components
and humoral factors of PRP vary significantly depending on the preparation methods used, and the leucocyte
content is used to categorize PRPs as pure PRP, leucocyte-poor PRP (LP-PRP), or leucocyte-rich PRP (LR-PRP).
More recently, autologous protein solution (APS), a version of LR-PRP with a higher concentration of humoral
factors, has received some attention.

1

Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, Isehara, Kanagawa,Japan. 2Center for Musculoskeletal innovative Research and Advancement (C-MiRA), Tokai University, Graduate
School of Medicine, Isehara, Japan. ✉e-mail: sato-m@is.icc.u-tokai.ac.jp

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Healthysubjects

OAK patientsgroup Z

group J

n

10

6

6

Female

5

5

4

Male

5

1

2

Age (years; mean ± standard deviation)

38.6 ± 11.0

72.5 ± 19.7

70.7 ± 8.1

2

2

0

3

3

5

4

1

1

K–L classification

Table 1. Characteristics of healthy subjects and OAK patients. K-L classification: Kellgren-Lawrenceclassification

WB
RBC (×104/μl)

APS

LP-PRP

HealthyGroup ZGroup J

HealthyGroup Z

HealthyGroup J

438.84 ± 46.44

402.60 ± 68.94

445.11 ± 76.44

250.67 ± 3.14

172.38 ± 61.96

3.14 ± 1.62

5.18 ± 7.74

PLT (×104/μl)

18.80 ± 5.99

18.47 ± 3.81

16.03 ± 3.26

127.03 ± 95.49

83.82 ± 54.34

95.49 ± 40.64

49.89 ± 17.19

WBC (×102/μl)

56.84 ± 20.25

48.90 ± 12.38

59.97 ± 6.38

530.01 ± 0.74

428.88 ± 138.06

0.74 ± 0.76

28.38 ± 54.82

PLT
concentration
ratio (%)

667 ± 342

495 ± 356

529 ± 256308 ± 129WBC
concentration
ratio (%)

963 ± 388

911 ± 319

1.54 ± 2.11

50.0 ± 86.0

Table 2. Blood cell analysis of each group. Whole blood (WB), APS, LP-PRP. Data reported as mean ± standard
deviation.

Recent studies have investigated the humoral factors associated with OAK, including those present in PRPs
prepared from healthy donors and/or from OAK patients of European or American descent8. However, to ourknowledge, there have been no previous reports concerning the humoral factors contained in PRPs preparedfrom healthy or OAK patients of Asian descent.
At Tokai University, we have begun to offer PRP treatment for knee disorders as a non-insured medical treatment approved under a provisional plan for class II regenerative medicine in accordance with the Act to Ensure
the Safety of Regenerative Medicine in Japan. We have offered two types of PRP: that prepared using the APS
kit (Zimmer Biomet, Warsaw, IN, USA), and that prepared using the Cellaid Serum Collection Set P type (JMS,
Hiroshima, Japan), categorized as LR-PRP and LP-PRP, respectively. In July 2018, we conducted a test run using
healthy subjects, and in September 2018, we began offering PRP treatment to OAK patients.
In the present study, we investigated the differences in the humoral factors and blood components between
PRPs prepared from healthy subjects and OAK patients using the two kits. The relationship between the levels ofhumoral factors and clinical outcome scores was also investigated.
This study was reviewed and approved by the Institutional Review Board of the Tokai University Schoolof Medicine (18R-134) and conducted in compliance with relevant guidelines. Written informed consent was
obtained from all participants.

Results

The characteristics of 10 healthy subjects and 12 OAK patients are shown in Table 1. There were no significant
differences in the characteristics of the two groups of OAK patients.

Blood cell analysis. The whole blood total cell count did not differ significantly between healthy subjects
and OAK patients (either group Z or J). We also compared the levels of red blood cells (RBC), platelets (PLT) andwhite blood cells (WBC) contained in PRPs prepared by the different kits or from healthy subjects compared with
OAK patients. Comparison of the kits indicated that RBC and WBC were significantly higher in group Z than ingroup J for both healthy subjects and OAK patients, but PLT did not differ significantly (Table 2).
The increase in concentration of PLT in the PRPs compared with that in whole blood was slightly higher forgroup Z than for group J in both healthy subjects (group Z: 667 ± 342%; group J: 529 ± 256%) and OAK patients(group Z: 495 ± 356%; group J: 308 ± 129%), but the difference was not significant.
The ratio of WBC concentration in PRPs to that in whole blood was significantly higher in group Z than ingroup J, although the WBC concentration was lower in group J for both healthy subjects (group Z: 963 ± 388%;
group J: 1.54 ± 2.11%) and OAK patients (group Z: 911 ± 319%; group J: 50.0 ± 86.0%).
Analysis of humoral factors. The comparison between kits showed that in healthy subjects, the concentrations of interleukin (IL)-1β, IL-1 receptor 2 (IL-1R2), IL-1 receptor antagonist (IL-1RA), tumour necrosis factor(TNF)-α, hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), soluble TNF-receptor 1

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Figure 1. Comparison of humoral factors in PRPs from groups Z and J of healthy subjects. (a–e) The
concentration of humoral factors in PRPs from groups Z and J of healthy subjects. (f–j) The total amount ofhumoral factors in PRPs from groups Z and J of healthy subjects. *P < 0.05, **P < 0.01. ND, non-detectableconcentration of humoral factors using ELISA. Values above the upper detection limit were not included in thecalculations, and the adjusted n numbers are as follows: TIMP-1 of group Z, n = 2; TIMP-1 of group J, n = 3.

(sTNF-R1), sTNF-R2 and soluble Fas (sFas) were all significantly higher in group Z than in group J. When the
total amounts of each humoral factor contained in the PRPs were calculated, the total amounts of IL-1β, IL-1RA
and HGF were significantly higher in group Z, and the total amounts of IL-1R2, TNF-α, PDGF, FGF, sTNF-R2,
sFas and TGF-β1 were significantly higher in group J (Fig. 1). In OAK patients, the concentrations of IL-1RA,
HGF, sTNF-R1 and matrix metalloproteinase (MMP)-13 were significantly higher in group Z, and only IL-1α
was significantly higher in group J. However, the total amount of IL-1RA and MMP-13 contained in the APSof group Z was significantly higher than that in group J, whereas the total amounts of IL-1α, TNF-α, TNF-β,
platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), sTNF-R2, sFas and transforming growth
factor (TGF)-β1 were higher in group J (Fig. 2).
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Figure 2. Comparison of humoral factors in PRPs produced from groups Z and J of OAK patients. (a–e)
Concentration of humoral factors in PRPs from groups Z and J of OAK patients. (f–j) Total amount of humoral
factors in PRPs from groups Z and J of OAK patients. *P < 0.05, **P < 0.01. ND, non-detectable concentrationof humoral factors using ELISA. Values above the upper detection limit were not included in the calculations,and the adjusted n numbers are as follows: MMP-3 of group J, n = 5.

The separate comparisons between healthy subjects and OAK patients of groups Z and J indicated that there
were significant differences between controls and patients of group Z in the concentrations of TNF-α, TNF-β,
VEGF, PDGF and MMP-13. The concentrations of TNF-α and PDGF were significantly higher in healthy subjects
than in OAK patients, and the concentrations of VEGF and MMP-13 were significantly higher in OAK patients(Fig. 3).
In group J, significant differences between controls and OAK patients were detected in the concentrations of
IL-1α, IL-1β, IL-6, TNF-α, TNF-β, VEGF, TNF-R2 and sFas: the concentration of TNF-α was higher in controls,
and the concentrations of the other factors were higher in OAK patients (Fig. 4).

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Figure 3. (a–e) Comparison of humoral factors in PRP prepared from healthy subjects and OAK patients ingroup Z. Significant differences were detected in the concentrations of TNF-α, TNF-β, PDGF, VEGF and MMP13. The plots show median values (middle lines), interquartile ranges (boxes), 1.5 IQR (whiskers) and outliers(dots). *P < 0.05, **P < 0.01 comparing healthy subjects and OAK patients.

Examination of clinical outcome scores.

The Knee injury and Osteoarthritis Outcome Score (KOOS)(symptoms, pain, activity, sports and quality of life [QOL] sub-scores and total scores) of both groups before PRP
treatment, and at 1, 3 and 6 months after treatment are presented in Figs. 5 and 6.
The symptoms sub-scores before PRP treatment differed significantly between groups, but there were no
significant differences in the other sub-scores or the total scores. However, for group Z, QOL sub-scores at 3
months improved significantly compared with the QOL sub-scores before PRP treatment (P = 0.007), while forgroup J, significant improvement was observed in the sports sub-scores between 1 and 6 months after treatment(P = 0.020).
The results of the analysis of the correlations between KOOS total score and either the concentration or
the total amount of humoral factors in PRPs are summarized in Table 3. Significant correlations were detected
between the following: the KOOS total score at 1 month after treatment and the concentrations of IL-1β, TNF-α,sTNF-R1, IL-6 and sFas; the KOOS total score at 3 months and the concentration of PDGF; and the KOOS total
score before treatment and the total amount of PDGF.
In addition, Table 3 shows the coefficients of correlation between the magnitude of change in KOOS total
score and either the concentration or the total amount of humoral factors in PRPs. Significant correlations weredetected between the following: the magnitude of change in KOOS total score at 3 months and the concentrationsof IL-1RA and sTNF-R1; the magnitude of change in KOOS total score at 1 month and the total amounts of IL-1β
and HGF; and the magnitude of change in KOOS total score at 3 months and the total amounts of IL-1α, IL-1RA,
TNF-β, FGF and sFas. However, it should be noted that these correlations are not strong.

Discussion

Recent studies have reported associations between OAK and the levels of humoral factors such as the well-known
inflammatory cytokines IL-1β and TNF-α. The activation of the nuclear factor (NF)-κB pathway by these
inflammatory cytokines results in the production of MMPs and VEGF, initiating an inflammatory response.
This inflammatory response can lead to synovial inflammation and cartilage degradation9,10. By contrast, the

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Figure 4. (a–h) Comparison of humoral factors in PRP prepared from healthy subjects and OAK patientsin group J. Significant differences were detected in the concentrations of IL-1α, IL-1β, IL-6, TNF-α, TNF-β,
sFas, VEGF and sTNF-R2. The plots show median values (middle lines), interquartile ranges (boxes), 1.5 IQR(whiskers) and outliers (dots). *P < 0.05, **P < 0.01 comparing healthy subjects and OAK patients.

anti-inflammatory cytokines such as the receptor agonists IL-1RA, sTNF-R1, sTNF-R2 and growth factor TGF-β
are well known for their cartilage-repairing activity11,12.
APS, which is prepared by concentration and enrichment of PRP by the addition of polyacrylamide beads,
contains a higher concentration of humoral factors than LP-PRP13. In this study, APS prepared from both healthy
subjects and OAK patients (group Z) contained a higher concentration of both anti-inflammatory cytokines andinflammatory cytokines compared with the LP-PRP of group J. Nevertheless, analysis of the total amount of thesecytokines within APS or LP-PRP revealed that for both healthy subjects and OAK patients, the total amounts of
TNF-α, PDGF, FGF, sTNF-R2, sFas and TGF-β1 were actually higher in group J, while the total amount of IL-1RA
was higher in group Z. Thus, when evaluating the clinical efficacy of different types of PRP kits, it is important toconsider both the concentration and the total amount of the different humoral factors.
A comparison between the PRPs produced from healthy subjects and OAK patients using each kit revealed
that the levels of the inflammatory cytokine TNF-α were higher for healthy subjects, in contrast to previous
reports analysing those produced from individuals of Europe and the United States14. The blood concentrationof TNF-α has been reported to increase in dementia and type 2 diabetes, as well as with age15,16. The reason forthe higher concentration in the small number of relatively young healthy subjects included in the present study isunclear, but the finding indicates the need for further studies.
There was a high concentration of VEGF in the PRPs prepared from OAK patients using both kits. In OAK
patients, VEGF is a direct initiator of cartilage destruction and synovial inflammation, and our results suggestthat systemic concentrations of VEGF are higher in OAK patients than in controls. We have previously reported
that bevacizumab, an anti-VEGF antibody, has an inhibitory effect on OAK after both intravenous (systemic) andintra-articular (local) administration17,18. This suggests that the pathology of OAK is not only local to the knee
joint but is also a systemic disease.
Clinical outcome scores evaluated using KOOS revealed a significant difference between kits only for the preoperative symptoms sub-score; no differences were detected in other sub-scores or KOOS total scores. However,
the extent to which clinical outcome scores improved for other sub-scores and KOOS total scores over time wasgreater for group Z than for group J; of note, the magnitude of change in symptoms and QOL sub-scores and the
KOOS total score at 3 months were significantly greater in group Z.
Kon et al. reported that compared with saline injection, APS injection significantly improved the Western
Ontario and McMaster Universities Osteoarthritis Index (WOMAC) percentage pain score from baseline to 1
year after treatment, and currently, the recommended schedule of APS treatment is a single rather than multiple
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Figure 5. KOOS sub-scores and KOOS total score. Symptoms sub-score before PRP treatment differedsignificantly between groups Z and J. In group Z, the QOL sub-score at 3 months improved significantly
compared with the sub-score before PRP treatment (P = 0.007). In group J, sports sub-scores improved
significantly between 1 and 6 months after treatment (P = 0.020). The plots illustrate median values (middlelines), interquartile ranges (boxes), 1.5 IQR (whiskers) and outliers (dots). *indicates significant difference
between group Z and group J. †indicates difference between follow-up time points in each group. *P < 0.05,**P < 0.01, †P < 0.05, ††P < 0.01.

Figure 6. Magnitude of change from preoperative scores for KOOS sub-scores and KOOS total score.
Magnitudes of changes in symptoms sub-scores, QOL sub-scores and KOOS total scores at 3 months weresignificantly higher in group Z, while the magnitude of change for QOL sub-scores at 1 month was significantly
higher in group J. This figure shows mean ± standard deviation. *P < 0.05, **P < 0.01.

injections per year19. However, the present study showed that the magnitude of change in clinical outcome scorestended to be greatest at 3 months postoperatively, suggesting that APS injections every 3 to 6 months may beeffective. Future studies to confirm this will require a greater number of participants who undergo 12 months of
postoperative follow-up.

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KOOS total score

Magnitude of change in KOOS total score

Concentration
IL-1αTotal amountConcentration

Total amount

pre

1 mo

3 mo

6 mo

pre

1 mo

3 mo

6 mo

1 mo

3 mo

6 mo

1 mo

3 mo

6 mo

− 0.244−0.309−0.595−0.622−0.0480.073−0.536−0.483−0.307−0.629−0.6040.000−0.705*−0.536IL-1β− 0.168−0.649*

0.281

0.231

−0.030−0.553

0.192

0.233

−0.530

0.516

0.340

−0.674*

0.283

0.287

IL-1R2− 0.194−0.222−0.192−0.013

0.221

0.402

−0.2120.186−0.258−0.096

0.038

0.070

−0.4690.089

IL-1RA− 0.080−0.323

0.464

0.247

−0.058−0.306

0.470

0.253

−0.1330.689*

0.290−0.1440.679*

0.294

TNF-α

− 0.513−0.634*−0.268−0.389

0.064

0.248

−0.371−0.267−0.3620.069−0.2390.037−0.550−0.356

TNF-β− 0.387−0.080−0.401−0.504

0.084

0.466

−0.389−0.2080.329−0.215−0.3720.381−0.592*−0.251

HGF− 0.297−0.5150.030−0.129−0.125−0.358−0.182−0.214−0.4360.288−0.047−0.601−0.140−0.241

PDGF

0.500

0.374

0.640*

0.094

0.581*

0.546

0.110

−0.007−0.0610.440−0.158−0.090−0.338−0.281

TIMP-1− 0.144−0.2440.208−0.223

0.159

0.153

−0.040−0.250−0.0780.399−0.152−0.067−0.186−0.290

VEGF− 0.075−0.208

0.288

0.025

0.101

0.187

−0.007−0.038−0.145

0.449

0.024

0.091

−0.094−0.093

FGF

0.481

0.296

0.245

0.340

0.430

0.422

−0.1750.013−0.178−0.0730.280−0.071−0.595*−0.073sTNF-R1−0.338−0.691*

0.200−0.069−0.018−0.217−0.211−0.350−0.469

0.550

0.032

−0.422−0.268−0.460sTNF-R2

0.220

0.042

0.436

−0.164

0.330

0.241

−0.031−0.298−0.1920.402−0.378−0.222−0.318−0.545−0.021

IL-6− 0.353−0.606*−0.133−0.099−0.130−0.335−0.259−0.119−0.467

0.116

0.079

−0.465−0.239sFAS−0.453−0.683*−0.204

0.028

0.049

0.092

−0.4070.007−0.404

0.105

0.294

−0.077−0.587*0.051

TGF-β1− 0.147−0.309−0.124−0.174

0.098

0.037

−0.166−0.077−0.359−0.043−0.123−0.221−0.305−0.121

MMP-3− 0.398−0.433−0.007

0.096

0.006

0.295

−0.222

0.092

0.111

0.369

0.365

0.470

−0.286

0.189

Table 3. Correlations between KOOS total score or magnitude of change in KOOS total score and theconcentration or total amount of the 17 different humoral factors measured in PRPs. The correlation coefficients(r) between the two parameters are shown in this table. *P < 0.05, **P < 0.01. Correlations were analysed forscores before PRP treatment and those at 1, 3, and 6 months after PRP treatment.

To examine the therapeutic effect of the concentration and total amount of humoral factors, we analysed thecorrelation between the KOOS and the concentration or the total amount of humoral factors contained in each
PRP. The concentrations of six humoral factors and the total amount of one humoral factor were correlated with
the KOOS total score, while the total amounts of six humoral factors and the concentration of one humoral factor
were correlated with the magnitude of change in the KOOS total score.
Both the concentration and total amount of PDGF were positively correlated with KOOS total score, but notwith the magnitude of change in the KOOS total score. This result suggests that the amount of PDGF contained in
PRPs tends to be higher in patients with higher KOOS, but this may depend mainly on other factors such as age.
Thus, a high level of PDGF may not necessarily be useful to predict the therapeutic effect of PRPs.
No correlation with KOOS total score was found for IL-1RA, which has attracted attention as a majoranti-inflammatory cytokine present in APS that has a pain-suppressing effect, or for TGF-β1. However, boththe concentration and the total amount of IL-1RA were significantly correlated with the magnitude of change in
KOOS total score, and IL-1RA was the only factor that was positively correlated with the magnitude of change
in KOOS total score. These results suggest that IL-1RA contained in APS and LP-PRP may have a positive therapeutic effect. However, the characteristics of the patients who produce high levels of IL-1RA in PRPs are unclear.
The characteristics of responders and non-responders to IL-1RA are also unclear, and this warrants further
investigation.
Humoral factors IL-1α, IL-1β, TNF-β, FGF and sFas were negatively correlated with the magnitude of change
in the KOOS total score. Of note, IL-1β and sFas showed a significant negative correlation with both KOOS total
score and the magnitude of change in KOOS total score.
IL-1β, which has various catabolic effects on chondrocytes, is associated with the induction of inflammatory cytokines such as IL-6, TNF and nitric oxide, up-regulation of aggrecanases and MMPs, down-regulationof extracellular matrix synthesis by reducing proteoglycan and collagen and cartilage surface dedifferentiation
through various signalling pathways, including the NF-κB pathway10,11,20.
sFas is an apoptosis-related protein, and its blood concentration is known to be elevated in OAK and rheumatoid arthritis (RA) patients21. Similar results were obtained in our study. Cheng et al.22 reported that sFas is likely
to inhibit Fas-mediated apoptosis and promote an autoimmune response in a mouse model of RA. It has beensuggested that sFas accumulates in the joints of RA patients as a result of this inhibition of apoptosis, leading toprolonged inflammation and synovitis. sFas concentrations in the joints of OAK patients have been reported to
be lower than those in the joints of RA patients23, but similar mechanisms may be responsible for inflammation
in OAK patients.
As described above, IL-1β and sFas in PRPs have negative effects on clinical outcome scores, and our results
suggest that patients with more IL-1β and sFas in their PRPs may have poor therapeutic responses and tend tohave lower KOOS total scores.
The concentration of VEGF, which was higher in OAK patients than in healthy subjects, did not correlate with
KOOS total score or the magnitude of change in KOOS total score.
MMP-3 and MMP-13 are known to be elevated in the synovium, synovial fluid and blood serum of OAK
patients14,24,25, but in this study, the concentrations and total amounts of MMP-3 and MMP-13 contained in PRPs
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were not correlated with KOOS total score. It has previously been reported that WOMAC scores and concentration of MMP-13 in blood serum were weakly correlated, while WOMAC scores after TKA surgery were positively
correlated with the concentration of MMP-13 within the synovial fluid14. An analysis of the concentrations of
MMPs in synovial fluid prior to PRP treatment may be warranted. Furthermore, the levels of tissue inhibitor of
metallopeptidase (TIMP)-1, an inhibitor of MMPs, did not differ significantly between PRPs prepared using thetwo kits and was not correlated with KOOS total score. Consideration of the relative levels of TIMPs and MMPs
may be useful26.
Future studies should include a larger number of subjects and an analysis of the correlation of PRP contents
with the findings of magnetic resonance imaging (MRI) techniques such as the Magnetic Resonance Observationof Cartilage Repair Tissue scoring system and the modified Noyes classification system, to determine the best typeof PRP to use and the timing of treatment relative to the degree of OAK.

Conclusion

In group Z, both anti-inflammatory and inflammatory cytokines were highly enriched in APS, and while theconcentrations of TNF-α, PDGF, FGF, sTNF-R2, sFas and TGF-β1 were lower in group J than in group Z, thetotal amount was higher.
For the clinical outcome scores, there was no significant difference in the KOOS total scores of group Z andgroup J after PRP treatment. However, the magnitude of changes in symptoms and QOL sub-scores and KOOS
total score at 3 months was significantly higher in group Z, while only the magnitude of change in the QOL
sub-score at 1 month was significantly higher in group J.
There was a significant positive correlation between IL-1RA levels and the magnitude of change in the KOOS
total score, suggesting that IL-1RA may contribute to the improvement of knee-joint function.
Further analysis of the relationship of PRP content with MRI findings including more subjects is necessary.

Materials and Methods

Ten healthy subjects over the age of 20 without OAK were included in the test run conducted in July 2018
(Table 1). Two types of PRP were purified separately from each healthy subject using the APS kit (group Z) or the
Cellaid Serum Collection Set P type (group J).
Twelve OAK patients who received PRP treatment between September 2018 and August 2019 at Tokai
University Hospital were prospectively randomized into group Z or group J (Table 1). The inclusion criteria for
OAK patients were as follows: age ≥ 20 years and experiencing knee pain; and Kellgren-Lawrence (K-L) classification of ≥ 2.
The exclusion criteria were as follows: positive for Hepatitis B surface antigen, hepatitis C virus antibodies, human immunodeficiency virus antibodies and/or Treponema pallidum antibodies; diagnosed with cancer;
affected by a serious medical condition; using anti-cancer drugs, biological drugs or immunosuppressants; active
infection; and a history of drug hypersensitivity.

Purification of APS and LP-PRP.

To prepare APS, 60 ml of blood (5 ml of anticoagulant citrate dextrose
solution, formula A [ACD-A] + 55 ml of blood) was collected in a 60-ml syringe. The blood was injected into the
APS Separator (Zimmer Biomet), which was then centrifuged at 745 ×g (3200 rpm) for 15 min using a GPSIII
centrifuge. Six millilitres of the cell solution were extracted and transferred to an APS Concentrator (Zimmer
Biomet). The device was then centrifuged at 291 ×g (2000 rpm) for 2 min using the same centrifuge, and approximately 2.5 ml of APS was collected from the device.
To prepare LP-PRP, 60 ml of blood (5 ml of ACD-A + 55 ml of blood) was used to prepare 6 ml of LP-PRP
using three Cellaid Serum Collection Sets P type. The Cellaid Serum Collection Set P type has a structure inwhich the primary and secondary containers have a connecting tube. After putting the primary container and thesecondary container in separate empty 50 ml centrifuge tubes, 20 ml of blood from the 60 ml syringe was injected
into the primary container and centrifuged at 200 ×g for 15 min. The supernatant containing platelets and plasma
was transferred to the secondary container, and then centrifuged at 1200 ×g for 15 min. Finally, excess plasma
in the secondary container was returned to the primary container, and 2 ml of LP-PRP was recovered from thesecondary container. The above procedure was repeated twice more to collect a total of approximately 6 ml of
LP-PRP.
From each healthy subject, approximately 120 ml of blood was collected using two 60 ml syringes, from which
2.5 ml of APS and 6 ml of LP-PRP were prepared.
From each OAK patient, approximately 60 ml of blood was collected using a 60 ml syringe; from this, 2.5 mlof APS from patients allocated to group Z and 6 ml of LP-PRP from those allocated to group J were prepared as
described above.

Blood cell analysis. Excess samples were collected from whole blood, plasma, APS and/or LP-PRP of eachhealthy subject and OAK patient, and blood cell analysis was performed to determine the number and concentration of RBC, PLT and WBC (Table 2).
One healthy subject and one OAK patient from group Z were excluded from the blood cell analysis because of
haemolysis and a lack of excess samples of APS, respectively.
Analysis of humoral factors. Using the excess samples of PRPs, catabolic factors (IL-1α, IL-1β, IL-6, IL-17,

TNF-α, TNF-β, MMP-3, MMP-13 and sFas), antagonists, inhibitors and decoy receptors of catabolic factors(IL-1RA, IL-1R2, sTNF-R1, sTNF-R2, and TIMP-1) and various growth factors (PDGF, FGF, TGF-β1, VEGF, and
HGF) were measured by enzyme-linked immunosorbent assays (ELISA).
IL-1RA and TGF-β1 were measured using the respective single-sample ELISA kits: Human IL-1RA
SimpleStep ELISA Kit (Abcam, Cambridge, MA, USA) and Human TGF-β1 Quantikine ELISA Kit (R&D
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Systems, Minneapolis, MN, USA). The other humoral factors were measured using Q-Plex ELISA multiplex
arrays (Quansys Biosciences, Logan, UT, USA). We analysed a total of 19 humoral factors and calculated theconcentration and total amount of each humoral factor in APS and LP-PRP.
The levels of humoral factors were compared between the PRPs produced by the two kits and between healthy
subjects and OAK patients for each kit. Values that were below the detection limit were treated as zero, and values
that were above the maximum detection limit were excluded (Figs. 1–4).

Examination of clinical outcome scores. The clinical outcomes of the 12 OAK patients were evaluatedby KOOS before PRP treatment and at 1, 3 and 6 months after treatment. The scores were compared over timeand between the two groups (Fig. 5).
In addition, the magnitude of change in KOOS from that before PRP treatment as baseline was calculated at 1,
3 and 6 months after PRP treatment, and compared between the two groups (Fig. 6).
Correlation coefficients were calculated to examine the correlation between KOOS and each humoral factor(Table 3). IL-17 and MMP-13, which were below the detection limit in six or more subjects, were excluded.
Statistical analysis.

The significance of differences between groups was determined using the Mann–
Whitney U test for non-normally distributed quantitative data. The Kruskal–Wallis test was used to detect significant differences between three groups or more.
Bivariate correlation analysis was performed using the Pearson’s test between KOOS and each humoral factor.
All statistical analyses were performed using SPSS software (v. 25.0; IBM, Armonk, NY, USA). P values of<0.05 were considered significant.

Received: 9 November 2019; Accepted: 2 June 2020;
Published: xx xx xxxx

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Acknowledgements

We thank Dr. Naoki Takatori, Dr. Toshihiro Noguchi, Dr. Yoshihiko Nakamura, Saori Shirasuna, and Ayako
Watanabe (Tokai University) for their clinical and technical contributions to this study.

Author contributions

S.W.: Designed the study, manufactured PRPs, analysed data and prepared the manuscript. M.S.: Guarantor,
designed the study, manufactured PRPs, analysed data and proofread the manuscript. M.M.: Designed thestudy, manufactured PRPs, analysed data and proofread the manuscript. E.T.: Designed the study, manufactured
PRPs and acquired data. R.U.: Manufactured PRPs and acquired data. T.T.: Acquired data and proofread themanuscript. E.O.: Manufactured PRPs and acquired data. Y.I.: Manufactured PRPs and acquired data. S.S.:
Manufactured PRPs and acquired data. M.W.: Designed the study and proofread the manuscript.

Competing interests

The authors declare no competing interests.

Additional information

Correspondence and requests for materials should be addressed to M.S.
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