Article

Combination Therapy of Autologous Protein Solution and
Extracorporeal Shock Wave Therapy for Severe
Knee Osteoarthritis
Shinya Nakasato 1,*, Tsukasa Kumai 2, Tomoki Mitsuoka 3, Takeshi Teramura 4 and Masao Akagi 3
Medical Corporation N Clinic, Osaka 596-0045, Japan
Faculty of Sport Sciences, Waseda University, Tokyo 169-8050, Japan; kumakumat@waseda.jp
3 Medical Corporation Honmachi N Clinic, Osaka 550-0005, Japan; mitsuoka@gaia.eonet.ne.jp (T.M.);
makagi@n-cli.com (M.A.)
4 Division of Cell Biology for Regenerative Medicine, Institute of Advanced Clinical Medicine,
Faculty of Medicine, Kinki University, Osaka 589-8511, Japan; teramura@med.kindai.ac.jp* Correspondence: nakasato@n-cli.com; Tel: +81-72-432-4976
1
2

Citation: Nakasato, S.; Kumai, T.;
Mitsuoka, T.; Teramura, T.; Akagi, M.
Combination Therapy of Autologous
Protein Solution and Extracorporeal
Shock Wave Therapy for Severe
Knee Osteoarthritis. Appl. Sci. 2024,
14, 1498. https://doi.org/10.3390/

Abstract: Knee osteoarthritis (OA) is a multifaceted metabolic disorder influenced by biomechanical, inflammatory, and immune system factors. Although autologous protein solution (APS) andextracorporeal shock wave therapy (ESWT) have shown promise for treating mild-to-moderate knee
OA, their eﬃcacy for severe cases remains limited when administered individually. Thus, we examined the combination eﬀects of APS and ESWT for severe knee OA. Twenty-four cases (33 knees) of
Kellgren–Lawrence grade 4 knee OA with bone marrow lesions and synovitis detected via magnetic
resonance imaging from December 2019 to November 2022 were included. All patients underwent
an ESWT session before the APS injection. The study included 20 knees in the APS + ESWT group,
which underwent an average of 4.5 ESWT sessions, and 13 in the APS-alone group. We evaluatedboth groups’ Knee Injury and Osteoarthritis Outcome Scores (KOOSs) before the APS injection at 3
and 6 months. Additionally, we compared the mean KOOS changes between the APS + ESWT and
APS-alone groups at 3 months (Pre-3M) and from 3 to 6 months (3–6M). Pre-3M showed no significant diﬀerence in the KOOSs between the two groups; however, there was a significant change in
3–6M (p < 0.05). Combining APS therapy with ESWT in severe knee OA was more eﬀective anddurable than APS alone.
Keywords: autologous protein solution; combination therapy; extracorporeal shock wave therapy;
knee osteoarthritis

app14041498
Academic Editors: Gang Wei
and Rossella Bedini

1. Introduction

Received: 3 January 2024

Osteoarthritis (OA) is mainly considered a disease of older people, as over one-thirdof the population over 65 years old present OA in at least one joint [1]. However, factors
such as obesity, sports, genetic factors, work, previous injuries, or socioeconomic status
can influence osteoarthritic changes in joints [2,3] in younger populations. Knee osteoarthritis (KOA) was believed to be a mere cartilage degeneration disease caused by cartilage
wear; however, it is now considered a complex articular metabolic disease associated withbiomechanics, inflammation, and complex biological responses that aﬀect the immune
system (the cartilage, subchondral bone, and synovial membrane) [4,5].
In addition, during the process of inflammation in OA, inflammatory mediators stimulate chondrocytes to produce lysis enzymes, maintaining a vicious cycle consisting of
inflammation, enzymes, chondrolysis, and inflammation [6].
Hence, treatment for knee OA has aimed to improve the anatomical, metabolic, andphysiological abnormalities that cause a vicious cycle. There are several potential

Revised: 27 January 2024
Accepted: 8 February 2024
Published: 12 February 2024

Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
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license(https://creativecommons.org/licenses/by/4.0/).

Appl. Sci. 2024, 14, 1498. https://doi.org/10.3390/app14041498

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Appl. Sci. 2024, 14, 1498

2 of 13treatments for this disease, including surgical administration, prescription drugs, physicalactivity, and traditional and new physical therapy methods [7]. Various treatment methods are currently being attempted. Orthologics, such as platelet-rich plasma (PRP) therapy, mesenchymal stem cell (MSC) therapy, and extracorporeal shock wave therapy(ESWT), are among these new treatments that are gradually gaining attention [8]. In particular, PRP injections have consistently outperformed HA injections in treating knee osteoarthritis, oﬀering pain relief and improved function [9]. As a result, PRP is recommended for cases where HA has not been eﬀective.
According to a report by Aso et al. [10], OA pain in the knee is dominated by pain fromthe subchondral bone and synovitis. The presence of a bone marrow lesion (BML), which isa lesion of the subchondral bone, is a risk factor for the structural progression of the disease;the presence of synovitis and joint effusion are also pathologies closely related to pain anddisease progression. When considering the treatment of knee OA pain, it is important tounderstand the cause and pathology of pain and take an appropriate approach. In recent
years, magnetic resonance imaging (MRI) has been used to diagnose knee joint pain, and it
has become possible to observe BMLs in the subchondral bone or synovitis in detail. Therefore, the cause of knee OA pain can now be easily identified by MRI.
APS, which is a condensed and processed form of PRP, has been developed for OA. In
OA, the inflammatory cytokines interleukin (IL)-1β and tumor necrosis factor (TNF)-α exacerbate cartilage destruction and OA progression. APS contains components that block theaction of these inflammatory cytokines [11]. APS reportedly reduces knee OA pain and prevents OA progression [12]. It is effective in patients with synovitis-derived intra-articularhydrops and is recommended for patients with persistent intra-articular hydrops.
On the other hand, ESWT has become a new conservative treatment for OA in recent
years [8,11,13]. ESWT reduces pain and improves motor function. Gao et al. [14] and Sansone et al. [15] reported ESWT as an eﬀective and reliable non-invasive treatment that rapidly reduces BMLs on pain-related MRI and improves knee function. In 2018, Kang et al.
reported the first use of targeted ESWT for BMLs in knee OA [16]. This treatment method
is based on the speculation that physical stimulation of bone marrow induced by ESWT
may improve bone remodeling abnormalities and promote the healing of microfractures[16]. Bone and cartilage regeneration reduces inflammation, induces angiogenesis, stimulates stem cell activity, and improves tissue regeneration and healing [13]. In addition,
ESWT promotes osteoblast growth, diﬀerentiation, and transforming growth factor(TGF)-β1 expression. Furthermore, OA treatment reportedly controls subchondral bone
remodeling and improves the trabeculae [13].
We previously performed MRI on patients with knee OA whose symptoms (pain andswelling) did not improve even after intra-articular injection of hyaluronic acid. As ESWT
acts directly on the bone, it is eﬀective against BMLs of the subchondral bone [17]. Conversely, APS is absorbed through the synovial membrane and acts primarily on the synovial membrane; thus, it is an eﬀective treatment for synovitis. Therefore, ESWT is recommended for patients with BMLs based on MRI findings and APS therapy is recommended
for patients with synovitis and intra-articular hydrops.
Some patients had both BML and synovitis, and they were treated with a combination of APS and ESWT to improve their condition. For the first year, we used both APS
and PRP, but APS therapy was overwhelmingly better at improving intra-articular edema,
and thus, we now recommend APS therapy to most patients, except for those whostrongly hope to receive PRP.
There are three reasons why we used a combination of APS and ESWT for severe
knee OA.
First, we thought that a single treatment would not be sufficient to deal with both synovial pain and subchondral bone pain, which are the causes of knee pain. In other words,
APS is effective for synovitis, but ESWT is effective for BML, and thus, we thought that severe knee OA with both synovitis and BML must be treated with both APS and ESWT.

Appl. Sci. 2024, 14, 1498

3 of 13

Second, many cases of severe knee OA, such as Kellgren–Lawrence classification
grade 4 (KL4), have fewer synovitis symptoms, such as joint fluid, which may reduce theeﬀectiveness of APS than KL2 or KL3. This was because we thought that applying somekind of stimulus to ESWT would make it easier for APS to respond.
Third, for severe KOA, such as KL4, there are some reports that ESWT and APS alone
were inefficient, and thus, we thought that it would be better if we combined both of them.
In 2013, Zhao et al. reported that ESWT was effective for KL2 and KL3 knee OA [8]. Moreover, in 2022, Ishihara et al. reported that APS therapy was administered for KL4 knee OA;
the effectiveness rate was 47.4% after 6 months of follow-up [18]. In addition, Nakajima et
al. performed APS on KL4 knee OA and reported that a few cases improved after 3 months[19]. APS and ESWT alone were reported to be effective for mild-to-moderate knee OA, suchas KL2 and KL3, but were less effective and short-lasting for more severe OA, such as KL4.
Kuwasawa et al. performed a 12-month follow-up study of 148 knees with KL2, 3, and 4 OA
that received APS injections and reported that the Knee Injury and Osteoarthritis Outcome
Score (KOOS) was significantly lower in KL4 than KL2 [20]. These reports showed that both
ESWT and APS were less effective for KL4 severe knee OA.
Since 2019, a combination of APS therapy and ESWT for knee OA has been used toincrease the success rate and duration in cases with synovitis and BML on MRI. The eﬀectiveness of these combination treatments for severe knee OA has not been reported.
Therefore, this study aimed to examine the eﬀects (success rate and duration) of a
combination of APS and ESWT on knee OA by severity.
2. Materials and Methods
2.1. Study Design
We used a retrospective study design. APS therapy was performed from December
2019 to November 2022 in patients diagnosed with knee OA at our clinic or another clinic.
Of the 39 patients (13 male individuals, 16 knees; 26 female individuals, 35 knees) who completed the Knee Injury and Osteoarthritis Outcome Score (KOOS) questionnaire at 3 and 6
months, 24 with KL4 (totaling 33 knees) were included. The KL classification was used todetermine the OA severity of the knee. The mean age of patients was 71.0 (52–89) years.
After the APS injection, the A + E group that underwent ESWT after APS therapy had
26 knees, with 1, 5, and 20 knees scored as KL2, KL3, and KL4, respectively. Those who
did not do ESWT after APS therapy (A-alone group) had 25 knees, with 6, 6, and 13 knees
scored as KL2, KL3, and KL4, respectively. In the revision, KL2 and KL3 were excluded
and only KL4 was included, which is more severe. As a result, the A + E group had 20
knees and the A-alone group had 13 knees. In the A + E group, an average of 4.5 ESWTs
were performed post-APS therapy (Table 1).
Table 1. Material. Inclusion criteria: The main inclusion criteria were being aged 50–90 years; KL
grade 4 medial KOA diagnosis on Rosenberg view radiographs; no improvement with conservative
treatment covered by insurance for at least 2 or 3 months (e.g., physical therapy, oral analgesics, andhyaluronic acid intra-articular injection); and MRI findings of BML, synovitis, and intra-articulareﬀusion. All patients agreed to pay for medical treatment not covered by public health insurance.
Exclusion criteria: Patients with rheumatoid arthritis or arthritis following other inflammatory or
metabolic diseases; history of cancer treatment, mental illness, other regenerative medical treatment(PRP and mesenchymal stem cell injection, among others); outer type or PF type OA; KL1–3 KOA;
history of in vitro shock wave treatment before receiving APS; and history of intra-joint injection of
HA after APS.

n
Age
Male, nFemale, n

APS Only
1374.0 ± 6.1
3
10

APS + ESWT
20
69.6 ± 9.4
7
13

All
33
71.0 ± 8.1
1023

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All patients underwent an ESWT session before the APS infusion and tried to continue ESWT once every 2 weeks for the first three times after APS therapy and once every
4–8 weeks thereafter. However, there were cases when patients did not attend the clinic
for social reasons. The group that underwent ESWT after APS therapy (A + E group) included 20 knees, averaging 4.5 ESWTs. The group that did not undergo ESWT after APS
therapy (group A-alone) had 13 knees. There was no significant diﬀerence in age or number of cases between the two groups. This study adhered to the tenets of the Declarationof Helsinki. Written informed consent was obtained from all patients.
2.2. ESWT Methodology
At our clinic, hyaluronic acid intra-articular injection therapy was performed under
the diagnosis of varus knee OA, but there was no improvement in symptoms, and ESWT
was indicated for patients with BML identified by MRI. The irradiation site was the medialcondyle of the femur and the medial plateau of the tibia, or both, where the BML wasobserved and the area where the pain was felt.
The shock wave was produced using a Duolith SD1 (Storz Medical, Tägerwilen, Switzerland). First, MRI confirmed the areas where the BML was found in the medial condyleof the femur and the medial plateau of the tibia. The patient’s knee was maximally flexed,
and the ultrasound probe was directed to the site where the BML was believed to be present to identify areas of cartilage thinning. The same part was considered to be the sitewhere the BML existed, and the position of the irradiation target was determined. The
operator’s free hand gripped the handpiece vertically against the target and began irradiation, gradually increasing the power level to the limit of the patient’s pain tolerance. The
maximum output was up to 0.25 mJ/mm2. Since the pain stimulus weakened and disappeared if the same area was irradiated continuously, we tried to irradiate the entire lesionin three dimensions from multiple directions while listening to the degree of pain of theexaminee in detail. As a general rule, ESWT should be irradiated at 2500 shots at 4 Hz atleast 3 times every 2 weeks. Afterward, if the improvement of symptoms was insuﬃcient,
irradiation was continued once every 2–4 weeks until the 3-month questionnaire.
2.3. APS Methodology
Intra-articular APS injections have been reported to improve pain and function inpatients with knee OA. APS is made by extracting growth factors and anti-inflammatory
molecules in PRP, including IL-1-receptor antagonist, soluble IL-1 receptor II, and soluble
tissue necrotic factor (TNF) receptor, through dehydration with polyacrylamide beads.
APS was prepared using the nSTRIDE APS Kit (Zimmer Biomet, Warsaw, IN, USA).
In the first step, 55 mL of blood and 4 mL of anticoagulant citrate dextrose solution A(Citra Labs, Braintree, MA, USA) were injected into the nSTRIDE Cell Separator, and approximately 6 mL of PRP was separated after centrifugation at 3200 rpm for 15 min. The
prepared PRP was then transferred to the nSTRIDE Concentrator, where it was exposed
to polyacrylamide beads and filtered by centrifugation at 2000 rpm for 2 min to produce
approximately 2–3 mL of APS. About 10 min after creating the APS, it was injected underultrasound guidance. If there was intra-articular eﬀusion after the joint puncture, joint
fluid was first aspirated as much as possible and then the APS was injected.
2.4. OutcomesFirst, we investigated the mean KOOSs before the APS injection, 3 months after the
APS injection, and 6 months after the APS injection in both the A + E and A-alone groups.
Next, to study the success rates, we examined the rates of 20% improvement and 50%
improvement in the KOOS in each group from the pre-APS score at 3 and 6 months.
The mean KOOS (improvement value [IV]) between the A + E and A-alone groups 3
months after APS injection (Pre-3M) and from 3 to 6 months (3–6M) were compared as astudy of the duration of eﬀects.

Appl. Sci. 2024, 14, 1498

5 of 132.5. Statistical Analyses
A paired t-test was used to compare the KOOSs before and 3 and 6 months after the
APS injection and before and after implementation, and an unpaired t-test was used forthe comparisons between the A and A + E groups. Statistical significance was set at p <0.05. Microsoft Excel 2016 (Microsoft, Redmond, WA, USA) was used for the analysis.
2.6. Ethical Procedures
Therapeutics performed in this study were reviewed and approved by the Technicaland Legal Advisory Committee for Regenerative Medicine of the Japan Advanced Medical
Association and received by the Ministry of Health, Labour and Welfare (approval no.
PB5180014) based on the Act on Securing Safety of Regenerative Medicine.
Informed consent to use the clinical data for study purposes was obtained from all
patients who were eligible for the treatments described in this paper.
3. Results (Table 2)
The average KOOS values were 39.5 ± 11.5 before the APS therapy, 51.6 ± 20.8 after 3
months, and 39.5 ± 20.5 after 6 months in the A-alone group. In contrast, in the A + E group,
the values were 45.0 ± 12.7 before the APS therapy, 52.5 ± 15.3 at 3 months, and 55.5 ± 13.2
after 6 months. The average KOOS decreased from 3 to 6 months in the A-alone group but
increased in the A + E group (Figure 1).
Table 2. Result (KOOS avg., KOOS pain, KOOS symp, KOOS ADL, KOOS sport, KOOS QOL).

Pre
A-alonePre
A+E
3 months
A-alone3 months
A+E
6 months
A-alone6 months
A+E

KOOS Avg.
(Figure 1)
39.5
±11.5
45.0
±12.7
51.6
±20.8
52.5
±15.3
39.5
±20.5
55.5
±13.2

KOOS Pain(Figure 2A)
46.0
±13.6
53.6
±16.3
59.6
±23.6
63.6
±19.0
44.0
±24.2
66.0
±12.8

KOOS Symp(Figure 2B)
47.6
±15.0
51.6
±13.1
63.2
±21.4
57.5
±17,9
46.9
±29.0
62.4
±11.3

KOOS ADL(Figure 2C)
57.7
±16.2
67.9
±15.1
65.5
±20.9
74.6
±17.8
55.6
±20.4
72.7
±15.7

KOOS Sport(Figure 2D)
24.3
±15.4
26.8
±14.3
38.9
±20.3
37.7
±15.7
26.0
±14.5
44.3
±21.9

KOOS QOL (Figure 2E)
40.4
±11.5
44.1
±12.7
51.6
±20.8
52.5
±15.3
39.5
±20.5
55.5
±13.2

The mean KOOS pain subscore was 46.0 ± 13.6 before the APS therapy, 59.6 ± 23.6
after 3 months, and 44.0 ± 24.2 after 6 months in the A-alone group. In contrast, in the A +E group, the values were 53.8 ± 16.3 before the APS therapy, 63.6 ± 19.0 at 3 months, and66.0 ± 12.8 after 6 months (Figure 2A). The mean other symptoms subscore was 47.6 ± 15.0
before the APS therapy, 63,2 ± 21.4 after 3 months, and 46.9 ± 29.0 after 6 months in the Aalone group. In contrast, in the A + E group, the values were 51.6 ± 13.1 before the APS
therapy, 57.5 ± 17.9 at 3 months, and 62.4 ± 11.3 after 6 months (Figure 2B). The mean
activities of daily living (ADL) subscore was 57.7 ± 16.2 before the APS therapy, 65.5 ± 20.9
after 3 months, and 55.6 ± 20.4 after 6 months in the A-alone group. In contrast, in the A +E group, the values were 67.9 ± 15.1 before the APS therapy, 74.6 ± 17.8 at 3 months, and72.7 ± 15.7 after 6 months (Figure 2C). The mean sports and recreation subscore was 24.3
± 30.9 before the APS therapy, 33.3 ± 32.1 after 3 months, and 25.0 ± 30.7 after 6 months in
the A-alone group. In contrast, in the A + E group, the values were 30.3 ± 25.7 before the
APS therapy, 34.8 ± 25.8 at 3 months, and 29.1 ± 24.0 after 6 months (Figure 2D). The meanquality of life (QOL) subscore was 22.8 ± 15.4 before the APS therapy, 38.9 ± 20.3 after 3
months, and 26.0 ± 14.5 after 6 months in the A-alone group. In contrast, in the A + E group,

Appl. Sci. 2024, 14, 1498

6 of 13the values were 26.8 ± 14.3 before the APS therapy, 37.7 ± 15.7 at 3 months, and 44.3 ± 21.9
after 6 months (Figure 2E). Most KOOS subscores decreased from 3 months to 6 months
in the A-alone group but increased in the A + E group.

Figure 1. The average KOOS decreased in the APS-alone group after 3–6 months but increased in
the APS + ESWT group. APS, autologous protein solution; ESWT, extracorporeal shock wave therapy; KOOS, Knee Injury and Osteoarthritis Outcome Score. The average KOOS decreased from 3 to
6 months in the A-alone group but increased in the A + E group.

Figure 2. The average KOOS subscores in the APS and APS + ESWT groups: (A) pain, (B) other symptoms, (C) activities of daily living (ADL), (D) sports and recreation, and (E) quality of life (QOL). APS,
autologous protein solution; AVG, average; ESWT, extracorporeal shock wave therapy. Most KOOS
subscores decreased from 3 months to 6 months in the A-alone group but increased in the A + E group.

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Regarding success rates, in the A-alone group, six patients (46.2%) had an improvement of ≥20% in their KOOSs after 3 months and five (38.5%) had an improvement of≥50%. In contrast, in the A + E group, 11 patients (55.0%) had an improvement of ≥20% intheir KOOSs and 4 (20.0%) had an improvement of ≥50% in their KOOSs. After 6 months,
two (28.6%) patients had an improvement of ≥20% in their KOOSs in the A-alone groupand one (14.3%) had an improvement of ≥50%. In the A + E group, seven (58.3%) patients
had an improvement of ≥20% in their KOOSs and five (35.7%) of ≥50%. The success ratein the A-alone group was higher at 3 months than at 6 months for both 50% and 20%
improvements. In the A + E groups, the success rate was higher at 6 months than at 3
months (Table 3).
Table 3. Success rates in A-alone and A + E groups. For both 20% and 50% improvements, group A
performed worse after 6 months than after 3 months, although group A + E performed better after
6 months than after 3 months.

Group A-alone, 3 months
Group A-alone, 6 months
Group A + E, 3 months
Group A + E 6 months

50% Improved
38.5%
14.3%
20.0%
35.7%

20% Improved
46.2%
28.6%
55.0%
58.3%

Regarding the duration, the IVs at Pre-3M were 12.1 ± 18.4 and 7.5 ± 12.6 in the Aalone and A + E groups, respectively, and −5.6 ± 8.1 and 5.1 ± 12.1 at 3–6M, respectively.
The mean KOOS at Pre-3M showed no significant diﬀerence in improvement between thetwo groups; however, the improvement was significant in the A + E group compared withthat in the A-alone group at 3–6M (unpaired t-test, p = 0.026; Figure 3). There were nocomplications or side eﬀects in the two study groups. Immediately after the APS injection,
there were cases of minor fever or swelling due to pain, which is common in the case of
APS only. As expected, the fever and swelling were reduced after a few days.

Figure 3. Mean improvement in the KOOSs between APS and APS + ESWT groups. The mean improvement value (IV) at Pre-3M showed no significant diﬀerence between the two groups; however,
the mean IV at 3–6M showed a significant diﬀerence between the two groups * p < 0.05). KOOS,
Knee Injury and Osteoarthritis Outcome Score; APS, autologous protein solution; ESWT, extracorporeal shock wave therapy; Pre-3M, before 3 months; 3–6M, from 3 to 6 months.

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4. Discussion
The mean KOOS of the A-alone group decreased between 3 and 6 months, and thesuccess rate in the A-alone group was higher at 3 months than at 6 months for both 50%
and 20% improvement in the A-alone group. These results suggest that the eﬀect of APS
therapy alone on severe knee OA, such as KL4, lasted for at most 3 months. This result is
consistent with those reported by Nakajima et al. [19].
However, the average KOOS in the A + E group increased between 3 and 6 months,
and the success rates in the A + E group were higher after 6 months than after 3 months
for both 50% and 20% improvement. These results suggest that ESWT after the APS injection improved the duration of the eﬀects and the performance rate.
In addition, the IV at 3–6M was significantly higher in the A + E group than in the Aalone group, suggesting that ESWT contributed significantly to improved performancefrom 3 to 6 months. Furthermore, the IV at 3–6M in the A + E group was 5.1 ± 12.1 comparedwith 7.5 ± 12.6 at Pre-3M; however, it was still a positive change. Further improvement could
be expected if irradiation continued after 6 months by adding ESWT. Therefore, additional
irradiation (booster irradiation) is considered effective in improving performance.
In more severe cases of KL4, the duration and success rate of ESWT after APS injection were higher than those of APS alone, suggesting that the combination of APS and
ESWT was more synergistic than ESWT or APS alone. However, the mechanism underlying the synergistic eﬀect remains unknown, although several hypotheses were proposed.
The mechanism can be inferred from highly severe knee OA characteristics, such as KL4.
According to Miller et al., increased pathological free nerve endings in knee joints
with advanced OA are important when considering the mechanism underlying chronic
pain generation [21]. In the present study, all patients were irradiated with ESWT once
before APS injection; however, in highly severe OA, such as KL4, BMLs are often enlarged
or advanced, and it is believed that there are more pathological free nerve endings. Consequently, a single irradiation was not suﬃcient, and it is thought that multiple additional
ESWT irradiations are required to destroy the free nerve endings present in BMLs. Additionally, as symptoms and living standards improve, activity levels naturally increase. OA
may worsen over time compared with before the APS injection. Therefore, multiple andadditional ESWT irradiations contribute to symptom or pathological improvement.
In addition, KL4 knee OA has less synovitis than KL2 and KL3, and fewer inflammatory cytokines are expressed [22]. APS targeting inflammatory cytokines may inevitably
be less eﬀective for severe knee OA, such as KL4. However, if ESWT stimulation increases
inflammatory cytokines, such as TNFα, even temporarily, it may make it easier for APS towork eﬀectively in response to TNFα [23]. Consequently, ESWT causes pseudo-inflammation, which facilitates the action of APS-containing anti-inflammatory cytokines. In addition, cell adhesion factors that have an aﬃnity for APS and PRP may be stimulated by
ESWT, facilitating the eﬀects of APS.
In addition, combined treatment may be expected to produce anti-inflammatory andcell proliferation eﬀects, as seen on radiographs of the KL4 front-of-knee OA of a 64-yearold woman before APS therapy and 48 months after APS therapy. This patient continued
ESWT approximately once or twice a month after the APS injection but showed a slight
opening of the joint dehiscence compared with that before the APS injection (Figure 4).
MRI of the same patient 48 months later showed the reduction in BML in T1 (upper photo),
and the disappearance of bone cysts in T2 (lower photo) was confirmed (Figure 5). Since
APS is a PRP product, it originally contains a large amount of TGFβ; however, the increased TGFβ level due to ESWT stimulation may have contributed to the anti-inflammatory eﬀect and the regulation of cell proliferation and diﬀerentiation [24,25].
If ESWT stimulates endogenous stem cells, the interaction between the stimulatedendogenous stem cells and the injected APS solution may promote cell proliferation[26,27]. However, further studies are required to confirm this hypothesis.

Appl. Sci. 2024, 14, 1498

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Figure 4. Radiography findings in the Rosenberg view showing KL4 medial knee OA in a 64-yearold female patient. (A) Joint space before APS. (B) Joint space is expanded 48 months after APS +ESWT. APS, autologous protein solution; ESWT, extracorporeal shock wave therapy; KL,
Kellgren–Lawrence classification; OA, osteoarthritis.

In addition, in severe knee OA, surgery, such as joint replacement, may be recommended, but it can be presented as a transition or alternative therapy for patients who cannot undergo surgery due to age restrictions or various social backgrounds. Regenerative
medicine for knee osteoarthritis has recently attracted attention, and orthologics, such as
PRP or MSC therapy, have attracted attention in recent years, but most of them are administered intra-articularly. When intra-articular administration is performed, most of the material is absorbed into the synovial membrane, except in severe cases of the rupture of somesubchondral bone plates. The synovial membrane nourishes the cartilage, but even if there
is an improvement in pain, it cannot directly improve the BML-derived pain from the bonemarrow. However, not all BML-induced bone pain is resolved with ESWT. In particular,
there seems to be a limit to pain with bone cysts and BML that contains a large amount of
osteonecrotic tissue. In addition, in BMLs where the subchondral bone plate is ruptured,
joint fluid easily enters the bone marrow and conservative treatment may be limited.
In this study, we recognize that further treatments other than ESWT that can repair
BMLs, bone cysts, necrotic tissues, or subchondral plates will be needed in the future.
This study had some limitations. First, the only endpoint was the KOOS, and thus, theresults are limited. The New Knee Society Score was to be used to evaluate patient-reported
outcome measures and objective knee joint function. However, studies in one clinic were limited in assessing the effect of treatment using the KOOS, which is most often used, from thepatient’s point of view. Second, image evaluation should not only be through KL classification
but it should also include the relationship with BMLs. In this study, ESWT was applied on
patients with knee OA with BMLs observed on MRI, but the type of BML irradiated and how
it is done should be determined. Third, it is unclear whether ESWT irradiation irradiates the
BML precisely or whether other areas are also irradiated. In addition, in this series, the numberof shock waves was set at 2500 per session, but the shock wave strength and number were notconstant and depended on the patient. While these were supposed to be constant, the shock
wave level chosen was the maximum strength the patient could tolerate; therefore, we had toobserve the patient’s reaction during and after the shock wave irradiation and determine thestrength and irradiation time of the shock wave. In addition, the number of irradiations was
the same regardless of whether there was one large BML or multiple, and thus, the energy

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irradiated per unit area varied depending on the size and number of BMLs. This was also
different from clinical trials, and it was a limitation of the actual treatment. Fourth, there weredifferences in how patients spent their time after APS or ESWT. It cannot be ruled out thatsome patients may worsen due to differences in life, work, or sports activities after APS or
ESWT. Unlike clinical trials, we believe that this is a limitation of research using actual patients.
Fifth, as these are case studies, they were not compared with controls. Sixth, a well-formulated
research plan was absent, but the APS treatments followed a research protocol. The decisionof whether to add shock waves may be easy in physical clinic visits.→Whether or not to do
ESWT depends on whether it is easy to go to my clinic. It is recommended that all patients
continue irradiation once every 2 weeks after the APS injection for a total of three times andonce every 4–8 weeks thereafter. However, whether to use ESWT after the APS injection wasdetermined by the patient. Some patients were subjected to additional shock wave irradiation
because their symptoms did not improve during treatment. However, some patients were notsubjected to additional irradiation because they felt unwell after the APS injection. We believethat these are the limitations of case reports rather than clinical trials.

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Figure 5. MRI findings of a KL4 64-year-old female patient. (A) BML and SBC on medial condyle
Pre-APS therapy. (B) In T1 (upper photo), MRI after 4 years confirmed a reduction in BML, and inT2 (lower photo), the disappearance of bone cysts was confirmed. 8 October 2019 MRI T1 Pre-APS
therapy (lt.) → 16 October 2023 MRI T1 4 years later after APS therapy (rt.); APS (31 October 2019)+ ESWT 36 times (every 1–2 months) after APS therapy. (A) coronal view (18 October 2019) (B) coronal view (16 October 2023) (C) sagittal view (18 October 2019) (D) sagittal view (16 October 2023).

5. Conclusions
This study revealed some interesting findings in assessing the KOOS before and 3
and 6 months after the APS injection. At the pre-APS injection and 3 months after the injection, no significant diﬀerence in the KOOSs was found between the A and A + E groups.
However, a noticeable and significant change was observed from 3–6 months later between the groups. This suggests that the combination of APS therapy and ESWT in severe
knee OA is more eﬀective and durable compared with APS alone. Furthermore, it wasAppl. Sci. 2024, 14, 1498

12 of 13suggested that in the treatment of knee OA, it is necessary to treat not only the synovitis
but also the subchondral bone.
Author Contributions: Conceptualization, S.N.; methodology, S.N.; software, S.N.; validation, T.K.,
M.A., T.T. and T.M.; formal analysis, M.A.; investigation, S.N.; resources, S.N., T.K., M.A., T.T. andT.M.; data curation, S.N.; writing—original draft preparation, S.N.; writing—review and editing,
S.N. and T.K.; visualization, S.N.; supervision, T.K. and M.A.; project administration, T.K. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Ethical procedures—therapeutics performed in this study
were reviewed and approved by the Technical and Legal Advisory Committee for Regenerative Medicine of the Japan Advanced Medical Association, and received by the Ministry of Health, Labour andWelfare (approval no. PB5180014) based on the Act on Securing Safety of Regenerative Medicine.
Informed Consent Statement: Informed consent to use the clinical data for study purposes was
obtained from all patients who were eligible for the treatments described in this paper.
Data Availability Statement: Data are contained within the article.
Conflicts of Interest: Authors Tomoki Mitsuoka and Masao Akagi was employed by the Medical
Corporation N Clinic and Medical Corporation Honmachi N Clinic. The remaining authors declarethat the research was conducted in the absence of any commercial or financial relationships that
could be construed as a potential conflict of interest.

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