Introduction
Osteoarthritis (OA) is the most common joint disease and a major cause of disability.
According to the National Health Interview Survey, more than 50 million adults have
been diagnosed with OA in the United States in 2012.[1] The knee is the one of the most commonly affected joint, and X-ray evidence of knee
OA is diagnosed in up to 60% of people older than 45 years.[2]
The etiology of OA is multifactorial and still not completely understood. Age, obesity,
lower-limb malalignment, cartilage defects, joint instability, previous fractures,
and meniscectomy surgery are all strongly correlated to knee OA.[3] Recently, literature reports suggested a female predisposition for developing knee
OA.[4] Overall, any condition that can cause articular damages, and all unfavorable biomechanical
conditions, which result in mechanical overload that exceeds the ability of a joint
to maintain itself, predisposes the knee to OA.[5]
During the past few decades, increased emphasis has been placed on the biochemical
balance required for the health of the cartilage. It has become evident that the inflammatory
mediators contribute significantly to the development and progression of structural
changes in the OA joint.[6] Because the induction of proinflammatory mediators in cartilage, synovial membrane,
and subchondral bone and their signaling pathways are interlinked and overlapped,
it therefore remains controversial whether inflammatory mediators are primary or secondary
regulators of cartilage damage and the defective repair mechanisms in OA.[7] Therefore, compounds that regulate cytokine and transglutaminases (TGs) synthesis
and activity are considered as favorable targets for future OA therapy. Recent research
focused on the anti-inflammatory effects of platelet-rich plasma (PRP). PRP is not
only a rich source of growth factors but also contains leukocytes, some residual erythrocytes,
metalloproteinases (MMPs), coagulation factors, and membrane glycoproteins. All these
components seem to be able to influence inflammation by regulating the synthesis of
other integrins, interleukins (ILs), chemokines, and cytokines.[8] However, PRP products can vary greatly, even in basic aspects such as their platelet
concentration or the content of white blood cells, and this may influence the results.
In vitro studies showed that PRP, in particular leukocyte-rich plasma (L-PRP), is
able to suppress inflammatory mediator concentration and gene expression in synovium
and cartilage tissue,[9] and this could explain the pain improvement and promising results in some clinical
series. Although there have been many clinical studies on the anti-inflammatory effects
of corticosteroids and hyaluronic acid (HA) injections for knee OA,[10] clinical studies using PRP are limited.
The aim of this study was to determine the clinical effect and outcomes of PRP intra-articular
injections for early stages of knee OA. The hypothesis of the study was that PRP can
become an important pain and inflammation mediator, especially in early stages of
knee OA.
Methods
Participants
Twenty-five patients matching strict inclusion and exclusion criteria received one
single PRP injection each for knee OA at our institution and were enrolled in this
study in 2014. The procedure was performed after the patients had signed a written
consent and the study was approved by the local ethics committee.
Inclusion criteria were patients affected by grade I and II knee primary OA according
to the Kellgren–Lawrence scale. Exclusion criteria were knee OA grade equal to or
greater than III, history of previous knee surgery, posttraumatic knee OA, and previous
infiltrative treatment of the affected knee. We also excluded patients affected by
rheumatic diseases, diabetes mellitus, and hematological disease (coagulation disorder).
Patients with platelet count of less than 150,000/mL were excluded from the treatment.
Radiographic examination performed prior to treatment (T0) included standard weight-bearing
X-ray of the affected knee in anteroposterior and laterolateral views and Rosenberg
view.
Interventions
PRP preparation was performed as follows. A 50-mL venous blood sample was collected
from each patient and centrifuged with GPS II system (Biomet Biologics, Warsaw, Indiana,
United States) for 15 minutes at a speed of 3,200 rpm. The mean volume obtained in
this series was 7.43 mL of PRP for intra-articular administration. The whole process
lasted approximately 20 minutes.
The patient was placed in a supine position with the knee in 90-degree flexion. The
skin was disinfected with alcohol or iodine-based antiseptic solution and draped.
PRP was injected very slowly in a sterile condition using a 22-gauge needle through
the anterolateral “soft spot.” Immediately after administration, gentle passive flexion
and extension exercises of the knee were encouraged. After the injection, the patients
were monitored for 10 minutes before discharge. The patients were advised to not take
any nonsteroidal anti-inflammatory drugs (NSAIDs) or to apply local ice for a week
after injection to avoid reduction efficacy on PRP. In the patients with bilateral
OA, both knees were injected. All knees received a single intra-articular PRP injection.
None of the patients received physical therapy after the injection.
Outcome Measurements
All patients were clinically examined on follow-up by a physician who was not involved
in PRP infiltration procedure. All the patients were evaluated using the Western Ontario
and McMaster Universities Osteoarthritis Index (WOMAC) and Knee injury and Osteoarthritis
Outcome Score (KOOS) scoring scales.[11]
[12] The questionnaires were administered at baseline (T0) and 6 months after PRP injection
(T1). Visual analog scale (VAS) score was recorded before treatment and at final follow-up.[13] Demographic characteristics of the patients as well as complications and adverse
events during treatment were recorded.
Data Analysis
Statistical analysis was performed using GraphPad Prism v 6.0 software (GraphPad Software
Inc.). Data were expressed as the mean ± standard deviation. D'Agostino–Pearson normality
test was used to test the normality of data distribution. Data were normally distributed,
and paired comparisons were performed by two-tailed paired t-test. The significance level was set at p-value lower than 0.05.
Results
Fifteen females (60%) and ten males (40%) with mean age of 49.7 years (range: 79–42
years) were enrolled. Preoperative plain radiographs showed grade I and grade II primary
OA in 8 and 17 patients, respectively. Of them, 21 were evaluated at after 6 months
(T1), whereas 4 patients were lost at final follow-up ([Fig. 1]). Mean WOMAC score at baseline was 29.1 ± 13.0, mean KOOS score before injection
was 37.5 ± 18.1, and mean VAS was 64.2 ± 14.6. At 6 months follow-up, the mean WOMAC
score improved to 42.4 ± 12.5, and the mean KOOS score was 59.7 ± 17.13. Improvements
in WOMAC and KOOS score were statistically significant (p = 0.0004 and p < 0.0001). We also found a significant improvement of the mean VAS score (42.8 ±
10.5; p < 0.0001), with all but one patient reporting pain relief 6 months after the procedure.
We did not observe any adverse reactions or other possible serious complications such
as infection after PRP injection.
Fig. 1 Flow diagram of the study.
Discussion
The main finding of this study is that PRP injection is an effective and safe treatment
for low-grade knee OA in terms of improving function and reducing pain at short-term
follow-up. This contributes to achieve the established goals for the treatment of
knee OA, such as relieving pain, improving function, increasing quality of life, and,
finally, reducing disability.[14]
[15] A variety of modalities have been used in the treatment of knee OA, including both
conservative methods and surgical methods. PRP has showed promising results for the
treatment of various musculoskeletal injuries and has gained popularity in the treatment
of knee OA due to its ease of use, low cost, and minimally invasive nature.[16] However, the exact mechanisms underneath its efficacy still have to be fully understood,
and often its administration is suggested on empiric rather than scientifically validated
basis.
The etiopathogenesis of OA is complex and involves many mechanical and biochemical
processes. Aging is the most important single risk factor of OA, and any unfavorable
biomechanical environment results in mechanical demand that predispose to articular
cartilage damage.[3] However, OA is not related to only mechanical stress, but many cellular and biochemical
processes are also involved in its pathogenesis.[17] In normal conditions, cartilage extracellular matrix is in a dynamic equilibrium.
In particular, the balance between anabolic and catabolic activities of chondrocytes
maintains the structural and functional integrity of cartilage.[18] In OA, a deregulated balance between proteinases degrading the extracellular matrix
and their inhibitors may be responsible for cartilage degeneration.[19]
Histomorphometrical and biomolecular investigations showed an increase of inflammatory
mediators and of TG-2 expression in human and experimental OA.[20] TGs have been implicated in the formation and development of hard tissue, extracellular
matrix maturation, and mineralization in growth plate cartilage. They are also involved
in organogenesis, tissue repair, and many pathological process.[21] TG-2, also known as tissue TG, is implicated in joint tissue remodeling, with particular
reference to the interplay with inflammatory mediators of OA. The capacity of transamidation
by TG-2 to regulate activation of latent transforming growth factor beta (TGF-β) seems
to have a potential impact on the regulation of inflammatory response in osteoarthritic
tissues.[14] TG-2 is also able to activate the crystal-promoting factor TGF- β1, and an overexpression
of TGF-β1 was found in laboratory of OA models.[22] TGF-β1 seems to play an important role in maintaining chondrocyte hypertrophy, which
is typically present near sites of cartilage surface damage, and it has also been
recognized as a crucial factor in the process of osteophyte formation.[23] Finally, inflammatory mediators implicated in OA, including IL-1, stimulate chondrocyte
matrix calcification.[24] Others inflammatory mediators such as matrix MMP-1, IL-6, IL-8, and chemokine (C-C
motif) ligand 5 are present and significantly higher in the synovial fluid of patients
with OA compared with normal patients.[6] These data confirm the complex role of TG-2 and inflammatory mediators during osteoarthritic
joint tissue remodeling and may play an important role in the development of OA. However,
it is not well known that if they are starters or just the consequence of degenerative
process of articular cartilage, and certainly, they are not the only pathogenetic
mechanism in the osteoarthritic process.
Recent in vitro studies showed an anti-inflammatory effect of PRP.[9] PRP is not only a rich source of growth factors, but it also contains antibacterial
and fungicidal proteins, MMPs, coagulation factors, and membrane glycoproteins that
influence inflammation by inducing the synthesis of other integrins, ILs, chemokines,
and cytokines.[25] El-Sharkawy et al[26] used monocyte culture to assess cytokine and chemokine levels, as well as monocyte
chemotactic migration, in the presence and absence of PRP. They showed that monocyte
chemotactic protein-1, which is released by monocytes in response to proinflammatory
stimuli, was significantly decreased by PRP in monocyte culture compared with untreated
cells, suggesting that PRP can act as an anti-inflammatory agent by producing endogenous
anti-inflammatory factors and by affecting monocyte cytokine release. Mazzocca et
al[27] developed an in vitro study to assess the anti-inflammatory effects of PRP on stimulated
human umbilical vein endothelial cells either alone or in combination with the corticosteroid
or NSAIDs. The authors concluded that PRP reduces cellular inflammation compared with
control. However, only one study has directly addressed effects of PRP on chondrocytes.
Sundman et al[28] assessed the anti-inflammatory effects of PRP in an ex vivo coculture model for
OA using human cartilage and synovium and concluded that PRP can stimulate endogenous
HA synthesis while decreasing cartilage catabolism and that it can also act to suppress
inflammatory mediator concentration in synovium and cartilage tissue. A recent review
concluded that although the effectors mediating the beneficial effects of PRPs have
not been identified, PRP could act as an endogenous source of chondroprotection by
interfering with the early catabolic and inflammatory events and by subsequently promoting
anabolic responses.[29]
Another important fact to be considered, is that many PRP formulation are available
for clinical uses and products can vary greatly, and it may influences the efficacy
of treatment and the results of clinical trials. Ehrenfest et al[30] proposed a classification based on platelet, fibrin, and leukocyte concentration:
pure PRP (P-PRP), leukocyte- and platelet-rich plasma, pure platelet-rich fibrin,
and leukocyte- and platelet-rich fibrin. In particular, the content and different
concentration of leukocytes in PRP products may affect the anti-inflammatory effects
of PRP. We used L-PRP type because it has been identified to improve cellular chemotaxis,
proliferation and differentiation, angiogenesis, and production of extracellular matrix,
and also being responsible for stimulating defense mechanisms against infections.
The role of leucocytes in PRP is a controversial issue in literature, and it has not
been proven that taking leucocytes from a PRP sample could either benefit or result
better outcomes for the patient.
McCarrel et al[31] demonstrated using tendonlike cells harvested and cultured from horse flexor digitorum
superficialis tendons that an increase in leukocyte content of PRP products is positively
correlated with an increased expression of inflammatory cytokines and that platelet/leukocyte
ratio had no influence on this effect. Among PRP formulations, a further division
can be made between those activated ex vivo with thrombin and/or calcium and those
inactivated, which rely on in vivo activation through endogenous collagen.[32]
Several authors have noticed reduction in pain after PRP application as reported in
our study. However, an explanation of this phenomenon has not always been given. Crane
and Everts believe that serotonin release from activated platelets might be responsible
for decreased pain.[33] Except for the growth factors in the alpha-granules, large amounts of serotonin
are contained within the dense platelet granules.[34]
Clinical studies on the anti-inflammatory effects of PRP injections are limited. A
recent systematic review on the use of PRP in intra-articular knee injections for
OA has been published. Eight studies published between 2010 and 2013 were included,
and all of them showed the efficacy of PRP in improving function and quality of life
and in reducing pain.[35] When PRP injections were compared with HA, the results of the studies did not reveal
superiority of PRP. Cerza et al[36] in their randomized controlled trial compared 120 patients treated with PRP (60
patients) and HA (60 patients) at a final follow-up at 24 weeks. Each patient received
four intra-articular injections, and the authors found significantly better clinical
outcome in PRP group compared with HA group, with lower WOMAC scores. In contrast,
other authors did not find any statistically significant improvement with PRP compared
with HA.[7]
The long-term efficacy of PRP and the number of doses to administer is another debating
matter because all studies published in literature to date have a short-term follow-up
(maximum 2 years), and only one long-term study suggests that the benefits of PRP
are not sustainable.[37] We also choose to use a single L-PRP administration because there is evidence of
no significant differences between a single dose and multiple doses, and therefore
a less invasive treatment could be performed.[37] This should represent an advantage compared with three HA injections or more and
also in terms of cost-efficacy.
Therefore, authors agree that PRP could be an effective treatment in the short term
to improve patients' function, quality of life, and reduce pain,[38] as it is reflected in the results of this study. According to literature, results
are worse in patients with gonarthrosis greater than grade II.[16]
The most relevant and clinically significant findings of this study were a notable
improvement in symptoms related to knee OA process, especially pain, demonstrated
in the results of the VAS scale in this study, and an improvement in quality of life,
with a high rate of satisfaction as evidenced by the results of KOOS and WOMAC questionnaires.
Moreover, we proved that a single dose of L-PRP can achieve satisfactory results,
reducing procedure-related morbidity and costs in comparison to multiple doses schemes
that have been used traditionally in previous studies.
The advantage of this treatment is that even if the mechanisms leading to clinical
effects are still to be understood, current literature agrees on safety of PRP, with
no serious complications reported.[37] Minor adverse events associated with repeated intra-articular injections include
moderate pain, swelling, and mild effusion that lasted a few days,[3] as shown in this study, where no adverse effects were reported.[39]
We acknowledge the limitations of this study. One limitation of this study is a relatively
short follow-up that cannot assess long-term results of intra-articular knee PRP,
which would determine whether there is some influence on the progression of the ostheoarthritic
disease or just a relieving of symptoms. The small sample size is also another limitation
of the study. The absence of a control group does not allow to draw a clear superiority
of PRP injection compared with the other available treatments. Thus, knee OA is a
chronic disease, and long-term outcomes should be an important consideration in evaluating
new treatments. Therefore, our short-term follow-up (6 months) does not allow us to
state if these results are maintained along time and to draw definitive conclusions.
In conclusion, a single dose of PRP in patients with knee OA grade I or II is a safe
an effective treatment for managing the symptoms associated with this pathology, especially
pain, and achieving improvements in quality of life of patients.
