JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINERESEARCH ARTICLE
J Tissue Eng Regen Med 2017.
Published online in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2238

Platelet-activated serum might have a therapeutic effecton damaged articular cartilage
Munetaka Yokoyama1, Masato Sato1*, Yoshiki Tani1, Miyuki Yokoyama1, Mami Kokubo2,
Masayuki Yamato2, Teruo Okano2 and Joji Mochida11Department of Orthopaedic Surgery, Surgical Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa,259-1193, Japan
2
Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo
162-8666, Japan

Abstract
Platelet-activated serum (PAS) was collected from rabbits. This contains high concentrations of growth factors, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB, and transforming growth factor-beta (TGF-β).
PAS was injected into the knee joints of Japanese White rabbits subjected to anterior cruciate ligament transection (ACL-T) to
investigate its therapeutic effects on articular cartilage. The effect of Avastin (an anti-VEGF monoclonal antibody) on VEGF expression was also investigated. The levels of VEGF, PDGF-BB, and TGF-β in PAS, platelet-rich plasma (PRP) and autologous serumfrom untreated rabbits were analysed by enzyme-linked immunosorbent assays. The samples (n = 24 rabbits) were divided into
control (C), PAS (S), Avastin (A) and PAS + Avastin (S + A) treatment groups. Intra-articular injections were administeredweekly for 7 weeks after ACL-T, during which the weight distribution ratios of the damaged limbs were evaluated. Histologicalevaluation was performed 12 weeks after ACL-T using Mankin score. The VEGF, PDGF-BB and TGF-β expression levels were signiﬁcantly higher (P < 0.05) in the PAS than in the PRP or autologous serum samples. The weight distribution ratios of damaged
limbs improved signiﬁcantly after ACL-T in all treatment groups (P < 0.05). The proximal medial, distal medial and lateral aspects of joints in the treatment groups showed signiﬁcant differences in Mankin scores compared with controls (P < 0.05).
The damaged limb weight distribution ratios, Mankin scores and articular cartilage structure did not differ signiﬁcantly among
the three treatment groups, which all showed signiﬁcant improvements in structure compared with controls. Copyright © 2017
John Wiley & Sons, Ltd.
Received 16 November 2015; Revised 24 April 2016; Accepted 3 June 2016
Keywords platelet-activated serum; articular cartilage repair; Avastin; vascular endothelial growth factor; anterior cruciate ligamenttransection; surgical model

1. Introduction
Osteoarthritis (OA) is the most common joint disorder in
the USA, and 10% of men and 13% of women aged 60
years and over have its symptoms (Zhang and Jordan,
2010). Articular cartilage is avascular tissue that isnourished solely by synovial ﬂuid, so it has poor regenerative capacity (Paget, 1969). OA progresses along withtrauma and the aging process, and complaints of problems in the knee joints are associated with increased physical activity in people of all ages. Many treatment methods
have been used to control the progression of OA and improve both pain and functionality. Non-steroidal antiinﬂammatory drugs, steroids, acetaminophen, opiates
and other drugs have been used as analgesics, but thesecause many adverse reactions (Bradley et al., 1991;
Dieppe et al., 1993; Langman et al., 1994). Intra-articular
hyaluronic acid (HA) injections and other minimally invasive treatments have been designed to alleviate the pain
associated with degeneration of articular cartilage.
*Correspondence to: Masato Sato, Department of Orthopaedic Surgery, Surgical
Science, Tokai University School of Medicine, 143 Shimokasuya, Isehara,
Kanagawa 259-1193, Japan. E-mail: sato-m@is.icc.u-tokai.ac.jp

Copyright © 2017 John Wiley & Sons, Ltd.

Although these are effective in the short term, they are insufﬁcient for the treatment of chronic and serious forms of
OA (Arrich et al., 2005; Petrella and Petrella, 2006). The
2013 American Academy of Orthopaedic Surgeons(AAOS) guidelines (http://www.aaos.org/Research/guidelines/OAKSummaryofRecommendations.pdf) strongly discourage the use of intra-articular injections of HA,
glucosamine or chondroitin. Therefore, many surgicaltreatments including microfracture and mosaicplasty havebeen used to facilitate tissue regeneration. However, thewidespread implementation of these methods has beenhampered by observations of ossiﬁcation and ﬁbrosis at
the transplant sites (Steadman et al., 1999; Szerb et al.,2005). Sporadic reports on treatment methods utilizing
components taken from the patient’s own blood indicatethat this methodology can result in the effective repair oftissue damage (van Buul et al., 2011; Filardo et al., 2011;
Kon et al., 2010; Patel et al., 2013; Sanchez et al., 2003;
Wehling et al., 2007; Yoshioka et al., 2013). Of these,
methods using platelet-rich plasma (PRP) have long
been used in dentistry and oral surgery, and many basic
research studies and clinical applications have been reported in the ﬁeld of orthopaedics since the ﬁrst study
by Sanchez et al. (2003).

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Serum has been included as a component of culture
media for promoting the proliferation of cells in regenerative medicine. Currently, sera of animal origins are used insuch media. Believing that the use of autologous serum in
culture media would be safer, the JMS Corporation (Hiroshima, Japan) has developed serum collection kits,
CELLAID®. Here, rabbit serum obtained using
CELLAID® kits was used to investigate its effect on the repair of damaged knee cartilage. These kits facilitate thecollection of platelet-activated serum (PAS) from bloodusing a sterile and closed procedure. PAS includes highconcentrations of growth factors, such as transforming
growth factor-beta (TGF-β) and platelet-derived growth
factor (PDGF)-BB. The kits also enable the long-term preservation of collected serum samples (Figure 1; Table 1).
Here we investigated the effects of intra-articular injections of PAS on the histological repair of defects generated
in knee joint articular cartilage in Japanese White rabbits.
Angiogenesis in synovial tissues is also a cause of inﬂammation, ectopic ossiﬁcation and pain in cartilage tissues in patients with OA (Haywood et al., 2003).
Therefore, the suppression of angiogenesis can be therapeutic against OA. Nagai et al. (2010) reported that the intravenous injection of bevacizumab, a humanized antivascular endothelial growth factor (VEGF) monoclonal
antibody (trade name, Avastin; Roche/Genentech, SouthSan Francisco, CA, USA), facilitated the repair of articular

M. Yokoyama et al.
Table 1. Comparison between PAS, autologous serum and PRP
Autologous serum•Autologous serum conditioned•Anti-inﬂammatory•Inhibition of tissue degeneration•Competitive blockade of IL-1 receptors
PRP•Plasma with signiﬁcantly elevated thrombocyte concentration from venous blood•Activation of tissue regeneration by growth factors•Growth factors are continuously released after the activation of thrombocytes
PAS•Platelet is activated by platelet activation beads•Including higher concentrated growth factors than autologous serum and PRP•CELLAID® can be preserved at 4 °C or 80 °C to 20 °C•Platelet activation beads contained in CELLAID® complete all serum preparation
steps within 1 h
PAS contained higher concentrations of cell growth factors (VEGF, TGF-β and PDGF-BB;
released by shaking with platelet-activating beads) compared with autologous serum
and PRP. PAS could be separated from the blood components using a closed and sterile
procedure with CELLAID® kits, and could be cryopreserved at –80 °C for over 1 year without any loss in growth factor concentrations.
IL, interleukin; PAS, platelet-activated serum; PRP, platelet-rich plasma.

cartilage damage in the knee (Nagai et al., 2010). Furthermore, the intra-articular injection of Avastin in models of
OA produced by anterior cruciate ligament transection(ACL-T) was shown to induce articular cartilage repair(Nagai et al., 2014). We hypothesized that the effect of
Avastin on articular cartilage repair, combined with PAS
obtained using CELLAID® kits might inhibit exogenous
VEGF. Therefore, we also administered intra-articular injections of Avastin with and without PAS to test rabbits
subjected to ACL-T. This work has already been published

Figure 1. Speciﬁcations of CELLAID® kits. These allow the separation of serum containing a high concentration of cell growth factors, such as transforming growth factor-beta(TGF-β), vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF)-BB from blood components in a closed, sterile procedure. The growth factors are
released by shaking with platelet-activating beads. It also allows for long-term cryostorage of the collected samples. (a) Fresh blood is introduced into the collection container,and (b) shaken for at least 30 min at room temperature. (c) The platelet-activating beads agglutinate rapidly. Centrifugation at 2000 g for 7 min is used to separate the serum fromthe beads. (d) The serum is transferred to storage container. (e) The storage container can be preserved at 4 °C, –20 °C or –80 °C. A plastic catheter is used to withdraw serum fromthe port [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

Copyright © 2017 John Wiley & Sons, Ltd.

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PAS might have a therapeutic effect on damaged articular cartilage

as part of the conference proceedings for the 2014 Orthopaedic Research Society (ORS2014) in New Orleans(http://www.ors.org/Transactions/60/0355.pdf).

2. Materials and methods
2.1. Ethical approval
All animal experiments were approved by the Ethics Review
Board of Tokai University, and were performed in accordance with the Tokai University Guidelines on Animal Use.
2.2. Quantitative analysis of growth factors
Platelet-activated serum was collected from ﬁve JapaneseWhite rabbits aged 12 weeks (weight ~2.5 kg) using
CELLAID® kits; the serum was cryopreserved at –80 °C.
The levels of growth factors, including VEGF, PDGF-BB
and TGF-β, in PAS, PRP and autologous serum samples
obtained from two Japanese White rabbits (aged 12
weeks; weighing ~2.5 kg) were analysed using commercial enzyme-linked immunosorbent assay (ELISA) kits
(Quantikine®; R&D Systems, Minneapolis, MN, USA).
We collected PRP and autologous serum from two Japanese White rabbits (aged 12 weeks; weighing ~2.5 kg)
using the following methods.
For PRP, venous blood samples were taken with
anticoagulant-coated tubes to avoid platelet activationand degranulation. Samples were centrifuged at 1000rpm for 5 min. At the bottom of the tube, the packed erythrocytes typically constitute 55% of the total volume. At the
top of the tube, platelet-poor plasma (PPP) constitutes 40%of the volume. An intermediate layer of 5% is called the
buffy coat. We aspirated the PPP and buffy coat layers using
a sterile syringe. This was centrifuged at 1000 rpm for 5min, and we aspirated the upper layer to collect the PRP.
For autologous serum, venous blood samples weretaken with no anticoagulant. The tubes were shaken ﬁvetimes, kept for at least 30 min at room temperature, andthen the upper layers were aspirated.
2.3. Histological evaluation of cartilage repair
We investigated the effect of PAS on articular cartilage repair in 24 Japanese White rabbits aged 12 weeks, subjected to ACL-T in the right knees with the animals
under general anaesthesia. Based on the observations ofNagai et al. (2010, 2014), an Avastin intra-articular injection (25 mg/ml) group was included. Six rabbits (sixknees) were each assigned to intra-articular injections of
1 ml phosphate-buffered saline (PBS) alone as a control(C) group, a PAS (S; 0.5 ml PAS + 0.5 ml PBS) group,an Avastin (A; 0.5 ml Avastin + 0.5 ml PBS) group and a
PAS + Avastin (S + A; 0.5 ml PAS + 0.5 ml Avastin)group (n = 24 knees in all). The knees were injected with
1 ml of the treatment solutions in PBS weekly, from weeks
1–7 after ACL-T, and the weight distribution ratios of thedamaged knees were measured (see below). The animals
Copyright © 2017 John Wiley & Sons, Ltd.

were killed 12 weeks after surgery by an intravenous overdose of anaesthetic. Medial and lateral tissues from thefemoral and tibial ends of the right knees were collected
and ﬁxed and processed for histology, and subjected tohaematoxylin and eosin and Safranin O staining. Immunohistochemistry was performed as described (Nagai
et al., 2008). Brieﬂy, sections were deparafﬁnized according to standard procedures. The sections were treatedwith 0.005% proteinase (type XXIV; Sigma-Aldrich, St
Louis, MO, USA) for 30 min at 37 °C for antigen retrieval.
For types I and II collagen, a primary mouse monoclonal
antibody (Daiichi Fine Chemical, Toyama, Japan) diluted
1:200 in PBS + 1% bovine serum albumin (Sigma-Aldrich; ﬁnal concentration 2.5 mg/ml) was placed on thesection overnight at 4 °C. Blinded histological evaluationsof the prepared tissue samples were conducted independently by three researchers using the histological scoreof Mankin et al. (1971; Mankin score).
2.4. Pain evaluation
The weight distribution ratios of the damaged limbs were
measured using an incapacitance test meter (Linton Instrumentation, Norfolk, UK) during weeks 1–7 after surgery, and pain in the damaged limbs was evaluated using
the following formula: damaged limb weight distribution
ratio (%) = [weight exerted by the damaged limb/(thatof the damaged limb + that of a healthy limb)] × 100.
The incapacitance meter is a device that facilitates automatic and reproducible pain evaluation by measuring theweight distribution of both hind limbs using a dual channel weight-averaging technique. It is widely used to investigate pain-ameliorating effects (Ito et al., 2012).
2.5. Statistical analysis
Comparisons between groups were made using analysis ofvariance (ANOVA). Scheffe’s post hoc test was applied,and P < 0.05 was assumed to be statistically signiﬁcant.

3. Results
3.1. Quantitative analysis of growth factors
Analyses of the levels of VEGF, PDGF-BB and TGF-β
expressed in PAS, PRP and autologous serum obtained
from two rabbits measured by ELISA indicated signiﬁcant
increases in VEGF levels in PAS (117.5 pg/ml) and PRP(90.3 pg/ml) compared with autologous serum (72.5
pg/ml; Figure 2a). PDGF-BB and TGF-β levels were alsosigniﬁcantly higher in PAS than in PRP and in the autologous serum (Figure 2b and c).
3.2. Pain evaluation
As shown in Figure 3a, the weight distribution ratios ofdamaged limbs showed a signiﬁcant improvement in the
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M. Yokoyama et al.

Figure 2. Quantitative analysis of growth factors. Platelet-activated serum (PAS), platelet-rich plasma (PRP) and autologous serum samples were collected from two JapaneseWhite rabbits aged 12 weeks (weight ~2.5 kg). They were analysed for vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF)-BB and transforming
growth factor-beta (TGF-β) expression levels using ELISA kits. (a) Signiﬁcantly higher quantities of VEGF were observed in PAS and PRP than in the autologous serum samples.
(b) PDGF-BB expression was signiﬁcantly higher in PAS than in PRP and in the autologous serum samples, and the PDGF-BB level in PRP was signiﬁcantly higher than that in
autologous serum. (c) A higher TGF-β concentration was observed in PAS than in PRP and autologous serum samples. The vertical axis is in pg/ml; *P < 0.05 and **P < 0.01 [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

three treatment groups from 5 weeks after ACL-T. A comparison of the weight distribution ratios of the damaged
limbs in the treatment groups at 8 weeks after surgery
showed signiﬁcant improvements (46.3%, 49.5% and48.0% in the S, A and S + A groups, respectively) compared with the C group (39.3%; P < 0.05). However, nosigniﬁcant differences were observed among the threetreatment groups (Figure 3b).

3.3. Histological evaluation of repair tissue
Macroscopic examination of the tissue revealed redness
on the surface of the articular cartilage on both theproximal and distal aspects in the C group at 12 weeks after surgery. A gross examination of the damaged knee
joints in the C group did not reveal any spur formationor cartilage loss, while there was a tendency for swellingand elevated temperature at the damaged sites. The articular cartilage histology ﬁndings (Safranin O, and Col-1
and Col-2 immunostaining) 12 weeks after surgery are
shown in Figure 4a–d for the C, S, A and S + A groups, respectively. The upper row in Figure 4 shows the articularcartilage histology of damaged knees demonstrated by
Safranin O staining 12 weeks after surgery. The surface
layers of the joints in the C group showed irregularities,
and the articular cartilage exhibited weak staining(Figure 4a). However, strong staining was observed in

Figure 3. Pain evaluation using damaged limb weight distribution ratios (%). (a) The damaged limb weight distribution ratios improved 5 weeks after anterior cruciate ligamenttransection (ACL-T) surgery in the three treatment groups (S, A and S + A). (b) The damaged limb weight distribution ratios improved signiﬁcantly in the treatment groups 8
weeks after surgery, compared with the control (C) group (*P < 0.05) [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

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PAS might have a therapeutic effect on damaged articular cartilage

Figure 4. Histological ﬁndings of the developed repair tissue 12 weeks after surgery. C group: Safranin O staining, and Col-1 and Col-2 immunostaining of samples from the control group 12 weeks after surgery are shown. The C group samples exhibited articular surface layer irregularities and weak Safranin O staining of the articular cartilage. Both Col1 and Col-2 expression levels were low at sites of weak Safranin O staining. S, A S + A groups: Safranin O staining, Col-1 and Col-2 immunostaining of samples from the S, A and S+ A groups 12 weeks after surgery are shown. All three groups exhibited strong Safranin O staining of the articular cartilage. Col-2 was strongly expressed at sites of strong Safranin O staining, while Col-1 was only weakly expressed. The histological ﬁndings of the treatment groups did not differ signiﬁcantly from those of the control group [Colour
ﬁgure can be viewed at wileyonlinelibrary.com]

the treatment groups (Figure 4b–d). This suggested thatthe articular cartilage had degenerated in the C group.

3.4. Immunohistochemistry
The features of damaged knee tissues of the C, S, A and S+ A groups, observed 12 weeks after surgery by immunohistochemical staining for Col-1 and Col-2 are shown in
Figure 4a–d. Type II collagen (Col-2) expression suggests
the repair of damaged articular cartilage by hyaline cartilage, while type I collagen expression (Col-1) suggests repair by subchondral bone ﬁbrocartilage. We observed astrong type II collagen expression in the treatment groups
12 weeks after surgery. However, type I collagen was
rarely observed in these sites, despite the strong Safranin
O staining. Types I and II collagen were scarcely expressedin group C, accompanied by weak Safranin O staining.
This suggests that the articular surface had been repaired
effectively with hyaline cartilage in the treatment groups.

Table 2. Mankin scores
I. Structur
0: Normal
1: Surface irregularities
2: Pannus + irregularities
3: Clefts to transitional
4: Clefts to radial
5: Clefts to calciﬁed
6: Disorganization
II. Cells
0: Normal
1: Diffuse hypercellularity
2: Cloning
3: Hypocellularity
III. Safranin-O staining
0: Normal
1: Slight reduction
2: Moderate reduction
3: Severe reduction
4: No staining
IV. Tidemark
0: Normal
1: Vascularization
Mankin scores were used to evaluate cartilage repair histologically. The total potential
scores ranged from 0 to 14, with 0 indicating normal, 1–3 indicating mild degeneration,
4–7 indicating moderate degeneration, and ≥ 7 indicating severe degeneration.

3.5. Histological scoring of repair tissue
The articular cartilage in four sites (proximal medial,proximal lateral, distal medial and distal lateral) was evaluated histologically 12 weeks after surgery using Mankinscores (Table 2). Mankin scores are shown as the averageproximal medial, distal medial and distal lateral scores,
respectively. These were 7.2, 6.2 and 8.0 in the C group;
4.0, 3.2 and 3.7 in the S group; 2.7 for each site in the A
group; and 3.6, 2.2 and 3.2 in the S + A group (Figure 5a, c and d). These scores indicated signiﬁcant differences
between group C and the treatment groups (*P < 0.05,**P < 0.01 in Figure 5). The scores did not differ signiﬁcantly among the treatment groups. The average proximal
lateral Mankin scores did not differ signiﬁcantly among
Copyright © 2017 John Wiley & Sons, Ltd.

the groups: C, 4.5; S, 3.08; A, 3.2; and S + A, 2.8(Figure 5b).

4. Discussion
The early stages of OA are characterized by destruction of
hyaline cartilage and its extracellular matrix caused by release of the inﬂammatory cytokine interleukin (IL)-1(Goldring and Goldring, 2004; Rutgers et al., 2010; Smithet al., 1997). The production of intracellular inﬂammatory
substances promotes the breakdown of hyaline cartilage
matrix (Fukui et al., 2001; Goldring, 2000). There are
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M. Yokoyama et al.

Figure 5. Mankin scores 12 weeks after surgery. The articular cartilage repair at each site was evaluated 12 weeks after surgery using the Mankin scoring system. (a) The proximal medial scores differed signiﬁcantly between the C and the S, A and S + A groups. The treatment group scores did not differ signiﬁcantly between each other. (b) The proximal
lateral scores did not differ signiﬁcantly between the C, S, A and S + A groups. (c) The distal medial scores differed signiﬁcantly between group C and the S, A and S + A groups;
however, there were no signiﬁcant differences among the three treatment groups. (d) The distal lateral scores also differed signiﬁcantly between group C and the treatmentgroups, but there were no signiﬁcant differences among the latter. *P < 0.05 and **P < 0.01 [Colour ﬁgure can be viewed at wileyonlinelibrary.com]

new treatment options for patients with OA using components from their own blood, which facilitate histologicalrepair (Arend et al., 1994; Dinarello and Thompson,
1991; Granowitz et al., 1991; Polisson, 2001). One ofthese is PRP, a plasma layer containing a large amountof platelets, obtained by the centrifugal separation ofblood. The alpha granules within blood platelets containgrowth factors, such as PDGF, TGF-β1, ﬁbroblast growth
factor and VEGF. High concentrations of these growth factors are present in PRP (Kon et al., 2010). The injection of
PRP into a joint results in the binding of a high concentration of growth factors to cell receptors that might help
promote repair. Filardo et al. (2011) observed an early improvement in a group of patients with OA following theintra-articular injection of PRP.
Currently, serum (speciﬁcally animal-derived serum) is
frequently included in the culture media used in regenerative medicine to promote stem cell growth. CELLAID®
kits allow the collection of serum containing high concentrations of growth factors, such as TGF-β and PDGF-BB, tobe separated from blood components via the shaking ofplatelet-activating beads, and collected in a closed andsterile environment. They also enable the collected samples to be cryopreserved at –80 °C for a long time without
any loss in cell growth factor concentrations even after 1
year of storage (data not shown). The quantitative analysis of growth factors expressed in PAS collected using
CELLAID® kits showed high concentrations of VEGF,
PDGF-BB and TGF-β, compared with PRP or autologous
serum (Figure 2). However, tissue degeneration, accompanied by conditions such as ectopic ossiﬁcation, ﬁbrotic
degeneration and synovial hyperplasia, has been associated with angiogenic factors such as VEGF, which are
Copyright © 2017 John Wiley & Sons, Ltd.

present in large quantities in platelets (Alini et al., 1996;
Aoyama et al., 2004; Bluteau et al., 2007; Ludin et al.,2013). Moreover, high levels of VEGF have been reported
in chondrocytes from articular cartilage in rabbits with OA(Jansen et al., 2012). A previous report (Emami et al.,2012) also suggested that VEGF might inhibit cartilage repair, thereby contributing to the exacerbation of OA. However, Nagai et al. (2010) have reported that an
intravenous injection of Avastin induced effective repairof damaged articular cartilage. In another study comparing intravenous with intra-articular Avastin injections in
rabbits subjected to ACL-T, Nagai et al. (2014) reported
that intra-articular injections at approximately half thedosage used for intravenous injections suppressed ectopic
ossiﬁcation, ﬁbrotic degeneration, synovial hyperplasia
and pain. Emami et al. (2012) also reported that an
intra-articular injection of Avastin in a rabbit model effectively reduced ﬁbrotic degeneration.
We anticipated that there would be an increase in the
VEGF levels in the PAS used here as a result of platelet activation, and an inhibition in cartilage histological repair
caused by increased exogenous VEGF expression resulting
from intra-articular PAS injections. Therefore, we added agroup of rabbits including both Avastin and PAS treatments(S + A). Kon et al. (2010) and Wehling et al. (2007) havereported that PDGFs such as TGF-β and PDGF-BB serve as
cell growth factors that can effectively induce articular cartilage repair. Our comparison of autologous serum and PRP
with PAS revealed that the PAS contained high concentrations of TGF-β and PDGF-BB. This suggested that intraarticular injections of PAS might facilitate histological repair. Therefore, the S + A group was expected to exhibit
signiﬁcant repair compared with the control and other
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PAS might have a therapeutic effect on damaged articular cartilage

treatment groups, because of the effect of high densities ofgrowth factors and the anti-VEGF effect of Avastin. A comprehensive comparison of the pain scores, histological ﬁndings and histological scores of group C with the treatmentgroups 12 weeks after surgery indicated that there had
been cartilage repair in the treatment groups. However,the S + A group did not exhibit signiﬁcant repair compared with the other treatment groups. This was possibly
because a single dose of Avastin exerts only a limited inhibitory effect on VEGF. This suggests that repeated Avastin
doses administered over 1–2 weeks might provide optimal
effects (Chung et al., 2011; Emami et al., 2012). We believethat the doses and numbers of Avastin injections might nothave been sufﬁcient to inhibit the increased levels of exogenous VEGF produced by platelet activation. Nagai et al.
(2014) reported that elevated levels of matrix metalloproteinase (MMP)-3, MMP-13 and IL-1β detected 4 weeks after surgery were indicative of inﬂammation in synovial
tissues of models of early OA; however, they did not observe a signiﬁcant increase in VEGF levels.
In the present study, weekly intra-articular injections of
PAS and/or Avastin were administered at 1–7 weeks after
surgery, and the treatment outcome was assessed 12
weeks after surgery. VEGF levels in the synovial tissue begin to rise 4 weeks after surgery in conjunction with progression of OA, so a signiﬁcant treatment outcome
should have been exhibited in the S + A group by 12
weeks after surgery or by extending the period of PAS
and Avastin administration. However, increasing the
Avastin dosage and number of injections could lead to adverse drug reactions, such as haemorrhage, thrombosis,
proteinuria, delayed wound healing and hypertension(Hurwitz et al., 2004). The use of an intra-articular injection, as in the present study, is likely to minimize the riskof such adverse drug reactions (Nagai et al., 2014). Our
results suggest that the intra-articular injection of PAS at
the 12-week point (i.e. short-term progression of OA) facilitated articular cartilage repair.
Platelet-rich plasma has also been the subject of hundreds of publications in recent years. Protocols for PRP
preparation vary widely between authors and are often
not well documented in the literature. A PRP classiﬁcation
system is needed to more accurately compare protocols.
DeLong et al. (2012) suggested the ‘PAW’ classiﬁcation
system. The system is based on three components: (1)the absolute number of platelets; (2) the manner in whichplatelet activation occurs; and (3) the presence or absenceof leucocytes. In this study, we collected PAS using
CELLAID® kits. CELLAID® kits allow the separation ofserum containing a high concentration of cell growth factors, but the transfer to storage container is performed
with only eyeballing as a measuring tool. As a result, the
CELLAID® system cannot exclude the buffy coat layer,
which contains remained platelets and leucocytes.
Because manual processing is not a clearly deﬁned protocol, it might randomly lead to leucocyte-poor or pure
PRP or leucocyte-rich plasma and PRP (Ehrenfest et al.,2009). The effect of highly concentrated leucocytes
contained within PRP preparations has been debated.
Copyright © 2017 John Wiley & Sons, Ltd.

Sundman et al. (2011) suggested that leucocytes containand produce biologically active cytokines that are primarily catabolic or inﬂammatory and might inﬂuence the clinical outcome of PRP application. Delivery of concentrated
leucocytes to a site of injury might not provide afavourable environment for tissue repair or healing.
Platelets contain factors leading to increased anabolic
signalling; in contrast, leucocytes release catabolic signalling molecules. The optimal balance between anabolism
and catabolism for tissue repair and the concentration of
growth factors and cytokines required to maintain thisbalance remains unclear. Depending on the clinical application, preparations of PRP should be considered basedon their ability to concentrate platelets and leucocyteswith sensitivity to pathological conditions that will beneﬁtmost from increased platelets or reduced leucocyte concentrations. In addition, Sundman et al. (2014) suggested
that PRP treatment also resulted in a signiﬁcant reductionof MMP-13, an increase in HA synthase-2 expression insynoviocytes, and an increase in cartilage synthetic activity compared with HA. They also showed that PRP stimulated endogenous HA production and decreased cartilage
catabolism; in addition, PRP showed similar effects to HA
in the suppression of inﬂammatory mediator concentrations and expression of their genes in synoviocytes andcartilage. Boswell et al. (2013) suggested that increasing the platelet concentration within leucocyte-reduced
PRP preparations would result in the delivery of moreanabolic growth factors and less proinﬂammatory cytokines. Minimizing leucocyte numbers in PRP is more
important than maximizing platelet numbers with respect to decreasing the expression of proinﬂammatory
molecules and enhancing the expression of matrix molecules. As described above, it can be assumed that
leucocytes contained in PAS might have inhibited cartilage repair and inﬂuenced our results. In the preparation of PAS using CELLAID® kits, it would bedesirable to reduce the concentration of leucocytes.
In this study, the groups treated with PAS alone andAvastin alone both showed effective cartilage repair
compared with the control group. Although some of
the limitations of our study include deﬁciencies inselecting an optimal dosage, in the number of injections
and in the duration of administration of Avastin, thecombined administration of PAS and Avastin in the S+ A group did not show a therapeutic effect ondamaged articular cartilage by synergistic effects. In
addition, the mechanism of action of PAS remains
unknown. Although these ﬁndings must be veriﬁedcomprehensively in further studies, the administrationof PAS is expected to facilitate effective cartilage repair
in human patients with OA.

5. Conclusion
Intra-articular injections of PAS showed therapeutic effects
on cartilage histological repair and pain relief in a
J Tissue Eng Regen Med 2017.
DOI: 10.1002/termM. Yokoyama et al.

8Japanese White rabbit model of OA produced by ACL-T.
However, no synergistic effects of Avastin with PAS were
found.

Conﬂict of interest
We declare no conﬂicts of interest.

Acknowledgements
This work was supported by a Health Labor Sciences Research
Grant from the Ministry of Health, Labor and Welfare. The authors are grateful to the Education and Research Support Center,of Tokai University.

6. Author disclosure statement
MS received research funding from CellSeed Inc (Tokyo,
Japan).

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J Tissue Eng Regen Med 2017.
DOI: 10.1002/term