The aim of this study was to assess a novel approach to treating severe knee osteoarthritis by targeting synovial membrane, superficial articular cartilage, synovial fluid, and subchondral bone by combining intra-articular injections and intraosseous infiltrations of platelet rich plasma. We explored a new strategy consisting of intraosseous infiltrations of platelet rich plasma into the subchondral bone in combination with the conventional intra-articular injection in order to tackle several knee joint tissues simultaneously. We assessed the clinical outcomes through osteoarthritis outcome score (KOOS) and the inflammatory response by quantifying mesenchymal stem cells in synovial fluid. There was a significant pain reduction in the KOOS from baseline
Knee osteoarthritis (KOA) is a mechanically induced, cytokine and enzyme-mediated disorder comprising different phases and phenotypes, with pain as the clinical hallmark of the disease [
In patients with severe OA, the subchondral bone undergoes changes which include microcracks and structural defects, vascularization of channels, nerve growth, and a progressive replacement of the subchondral marrow with fibroneurovascular mesenchymal tissue changes which underpin the increasingly recognized crosstalk and pathway for direct transport of growth factors such as transforming growth factor B (TGF
As it is yet to be established which of the joint tissues or structures is the primary driver of KOA and therapeutic strategies that solely target one cell or tissue may well prove to fail, it is advisable that approaches to treating KOA should aim at reaching several joint tissues [
In patients with severe KOA, platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective, anabolic, anti-inflammatory, and immunomodulatory effects and to substantially reduce pain, relieve joint stiffness, and improve physical function [
The study was carried out in accordance with the international standard on clinical trials: Real Decreto 223/2004, Declaration of Helsinki in its latest revised version (Fortaleza, Brazil; 2013), and Good Clinical Practice Regulations (International Conference for Harmonization). The study protocol was reviewed and approved by the Reference Ethics Committee. All patients provided written informed consent before entry into the study.
Nineteen patients were initially assessed for eligibility. Patients were considered eligible if they were aged between 40 and 77 years and presented severe knee osteoarthritis according to radiographic confirmation (Ahlbäck degrees 3 and 4, on a scale from 1 to 4, with the highest degrees indicating more severe OA). Finally, 14 patients were enrolled in the study from January 2014. The inclusion and exclusion criteria that patients had to meet in order to be included in this study are as follows.
Inclusion criteria are the following: Patients of both sexes aged 40 to 77 years. Predominant internal tibiofemoral knee osteoarthritis. Joint pain above 2.5 VAS points. Radiographic severity degrees 3 and 4 according to Ahlbäck scale. Values of body mass index between 20 and 33. Possibility for observation during the follow-up period.
Exclusion criteria are the following: Bilateral knee osteoarthritis which requires infiltration in both knees. Values of body mass index > 33. Polyarticular disease diagnosed. Severe mechanical deformity (diaphyseal varus of 4° and valgus of 16°). Arthroscopy in the last year prior to treatment. Intra-articular infiltration of hyaluronic acid in the past 6 months. Systemic autoimmune rheumatic disease (connective tissue diseases and systemic necrotizing vasculitis). Poorly controlled diabetes mellitus (glycosylated hemoglobin above 9%). Blood disorders (thrombopathy, thrombocytopenia, and anemia with Hb < 9). Undergoing immunosuppressive therapy and/or warfarin. Treatment with corticosteroids during the 6 months prior to inclusion in the study.
The enrolment finished on 29 October 2014 and the pilot study was completed on 10 June 2015.
In the first visit, an orthopedic surgeon conducted a clinical and radiographic assessment of each patient, including their medical history and a complete blood count. Moreover, the doctor delivered a booklet that contained detailed instructions and the knee injury and osteoarthritis outcome score (KOOS) questionnaire, which had to be completed by the patients at the baseline visit and before follow-up visits. Patients were allowed to consume acetaminophen, but it was restricted 48 hours before filling the questionnaires.
Patients were identified by a code number and scheduled to undergo the experimental procedure, which consisted of three treatments of PRP on a weekly basis. The first treatment included one PRP intra-articular infiltration and two PRP intraosseous infiltrations (femoral condyle and tibial plateau). The next two treatments were conventional intra-articular injections.
90 mL of venous blood was extracted from the patient in order to prepare the PRP and withdrawn into 9 mL tubes containing 3.8% (wt/V) sodium citrate. Blood was centrifuged at 580 g for 8 minutes at room temperature. The 2 mL plasma fraction located just above the sedimented red blood cells, but not including the buffy coat, was collected in a tube and carried to the injection room for use. This plasma fraction preparation contained a moderate concentration of platelets (2 to 3 times the concentration of platelets compared with peripheral blood, depending on the platelet count and size as well as the hematocrit) and an absence of erythrocytes and leukocytes [
In the patient’s first treatment, one PRP intra-articular injection and two PRP intraosseous injections were performed. Under anesthesiologist surveillance, sedation of the patient was induced by infusing a single dose of midazolam (0.03–0.05 mg/kg) and fentanyl (3.2 mg/kg), in a peripheral vein; single or repeated dose of propofol was also administered (1-2 mg/kg), depending on the duration of the infiltration. The degree of sedation was −4 or −5 on Richmond Sedation Scale. The patient was positioned in a supine position on an operating room table and two marks were drawn in the medial region of the knee, one located 2 cm proximal and the other located 2 cm distal to medial joint line; the infiltration area was prepared with a povidone-iodine solution. Local anesthesia was conducted by injecting 2 mL of 2% mepivacaine into the periosteum of condyle and tibial plateau. After evacuating the totality of the synovial fluid, 8 mL of PRP (the first intra-articular infiltration of a series of three) was infiltrated intra-articularly through the mid-point area of the femoropatellar region using a lateral approach in order to reach the joint space after lateralization of the patella. Intraosseous infiltrations were performed with a 13 G trocar used for bone biopsy, which was manually introduced into the bone and inserted 2 cm into the medial tibial plateau and medial femoral condyle. Once the trocars were placed in the desired position, 5 mL of PRP was infiltrated into subchondral bone of each structure. The control of trocar placements was facilitated by using a fluoroscope (Figure
Fluoroscopic images. Intraosseous infiltration into the medial femoral condyle (a) and tibial plateau (b).
Two more intra-articular PRP infiltrations were performed 7 and 14 days after the first treatment. Moreover, the synovial fluid evacuated prior to the infiltrations was preserved for analysis.
Patients were called for follow-up visits 2 and 6 months after the last treatment visit in order to conduct clinical evaluation. During these visits, the patient submitted the questionnaires given at baseline. A rheumatologist carried out a clinical examination and an evaluation of pain and function by visual analogue scale (VAS) and Lequesne Index, respectively. Acetaminophen consumption was also controlled.
The primary outcome was defined as the decrease in knee pain from the baseline to second month and sixth month (endpoint), according to the KOOS questionnaire. Furthermore, measurement of VAS and Lequesne Index was also evaluated; the secondary outcomes included the other areas of KOOS: symptoms, function in daily living (ADL), function in sport and recreation (sport/rec.), and knee related quality of life (QOL).
To evaluate the safety of treatment, all complications and adverse events were assessed and reported during patient visits. Their nature, onset, duration, and severity were documented.
Presence of mesenchymal stem cells (MSC) in synovial fluids before and one week after intraosseous infiltration was evaluated by flow cytometry and cultures of colony-forming cells (CFU-F). Concerning flow cytometry, each sample was immunophenotyped using an 8-color direct immunofluorescence technique. Concentrated cell suspensions were stained with the following combination of monoclonal antibodies (MoAb) in order to detect the expression of CD105/CD45/CD73/CD271/CD34/CD13/CD90/CD44: [Brilliant violet (BV) 421/orange chrome (OC) 500/fluorescein isothiocyanate (FITC)/phycoerythrin (PE)/peridinin chlorophyll protein-cyanin 5.5 (PerCP-Cy5.5)/PE-cyanin 7 (PECy7)/allophycocyanin (APC)/APCH7]. Regarding CFU-F assay, collected synovial fluids were diluted in phosphate buffered saline (PBS) and centrifuged in order to harvest the cellular content. The sample was used for colony-forming assay (CFU-F) and seeded on a 100 mm diameter culture plate. Seven days later, plating colonies were noted and counted by 0.5% crystal violet staining.
Power analysis was conducted to estimate the minimum sample size needed to achieve 80% power at a 5% level of significance for the primary outcome measures. An assumed effect size of 10 points (minimal clinically important change, MIC) with a standard deviation (SD) of 12 points was used [
Demographic and medical variables (gender, age, and OA grade) were determined by the mean, standard deviation, range, and percent. For this study, a pair protocol analysis was used. Comparisons were performed by Student’s
A total of 19 patients were considered eligible to participate in this study, and 14 patients were finally enrolled (Figure
Enrolment and outcomes.
Nine of the thirteen patients who finished the study were men and four were women, with a mean age of
Demographic data and biological and clinical outcomes.
Demographic data | ||||||
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Patients | Total: |
Men: |
Women: |
Age: mean ± SD (range) | OA III: |
OA IV: |
13 | 9 (69.23) | 4 (30.77) | 62.23 ± 9.6 (47–75) | 9 (69.23) | 4 (30.77) | |
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Biological outcomes | ||||||
Baseline: mean ± SD | One week after infiltration: mean ± SD |
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MSC/ |
7.98 ± 8.21 | 4.04 ± 5.36 | 0.019 |
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CFU-F/mL | 601.75 ± 312.30 | 139.19 ± 123.61 | 0.012 |
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Clinical outcomes | ||||||
Baseline: mean ± SD | Endpoint: mean ± SD |
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Improved patients: |
Patients with MCII [ |
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KOOS pain | 61.55 ± 14.11 | 74.60 ± 19.19 | 0.008 |
13.10 ± 14.89 (24.19 ± 40.07) | 11 (84.62) | 8 (61.53) |
KOOS symptoms | 60.56 ± 17.35 | 71.70 ± 18.82 | 0.004 |
11.14 ± 11.34 (19.73 ± 25.42) | 11 (84.62) | 8 (61.53) |
KOOS ADL | 68.44 ± 14.08 | 80.86 ± 15.58 | 0.022 |
12.45 ± 17.31 (23.25 ± 38.82) | 11 (84.62) | 8 (61.53) |
KOOS sport/rec. | 29.23 ± 20.29 | 45.38 ± 22.40 | 0.017 |
11.78 ± 11.54 (76.94 ± 115.23) | 10 (76.92) | 7 (53.84) |
KOOS QOL | 28.10 ± 19.75 | 39.28 ± 16.52 | 0.012 |
14.90 ± 22.03 (66.66 ± 72.64) | 11 (84.62) | 8 (61.53) |
VAS | 6.77 ± 1.75 | 2.88 ± 2.48 | <0.001 |
−3.88 ± 2.82 (−55.04 ± 38.21) | 11 (84.62) | 10 (76.92) |
Lequesne Index | 8.69 ± 2.65 | 5.77 ± 3.49 | 0.008 |
−2.92 ± 3.35 (−31.18 ± 46.61) | 10 (76.92) |
OA: osteoarthritis; MSC: mesenchymal stem cells; CFU-F: cultures of colony-forming cells; VAS: visual analogue scale; KOOS: knee injury and osteoarthritis outcome score; ADL: function in daily living; sport/rec.: function in sport and recreation; QOL: quality of life;
Table
Regarding secondary outcomes, there was also a statistically significant improvement in all other areas of the KOOS (symptoms,
The improvement of the patients was observed at 8 weeks of follow-up, and it was maintained until week 24, when the study ended (Figure
Clinical outcomes. KOOS (a), VAS (b), and Lequesne Index (c) at baseline, 8 weeks after treatment, and 24 months after treatment. ADL: function in daily living; sport/rec.: function in sport and recreation; QOL: quality of life.
Two patients reported 2 adverse events likely unrelated to the treatment. One of the patients experienced an episode of fever associated with flu episode, and the other reported exacerbation of knee pain three months after the treatment. Both events were mended satisfactorily by oral pharmacological treatment, which was allowed in the study. In addition, one patient was excluded because of a popliteal cyst caused by sports activity which was treated with fluid drainage and corticosteroid infiltration.
Baseline levels of mesenchymal stem cells (MSCs) presented in synovial fluid were
Concerning cultures of colony-forming cells (CFU-F), a substantial reduction in the number of CFU-F was also observed one week after infiltration, namely, the number of CFU-F/mL before and after treatment of
The combination of intra-articular and intraosseous injections of PRP is an
Mechanisms of intra-articular and intraosseous injections of platelet rich plasma. Depiction of a new strategy to treat severe knee OA by targeting different knee joint structures such as synovial membrane (SM), synovial fluid (SF), articular cartilage (AC) with noncalcified cartilage (NCC) and calcified cartilage (CC), and subchondral bone (SB) with intra-articular injections (IA) and intraosseous infiltrations (IO) of platelet rich plasma (PRP) [
There are several potential mechanisms by which intra-articular injections and intraosseous infiltrations of PRP might reduce knee pain.
In this study, patients also showed a significant improvement in the secondary efficacy outcomes such as function in daily living (ADL), function in sport and recreation (sport/rec.), and knee related quality of life (QOL). This increased intolerable physical load might entail a positive chondroprotective and anti-inflammatory effect, since as several lines of evidence suggest, moderate mechanical loading of joints prevents cartilage degradation by suppressing the activation of NF
The significant reduction of MSC in SF after treatment with this novel PRP therapy is open to interpretation. Several studies have reported that the accumulation of MSCs in SF increases with the severity of osteoarthritis, joint damage, and the disease duration [
This study has some limitations. First, a relatively small number of patients were enrolled in the study with no control group, all belonging to the same severe KOA phenotype stage. Second, the clinical follow-up of 6 months seems to be a short period to draw conclusive clinical indications. Third, an evaluation of patients with X-ray or MRI has been very useful to document eventual changes in the subchondral bone after PRP treatment. Finally, a mechanistic account of the significant pain and SF MSCs reduction experienced by the majority of patients is lacking. The first three limitations are inherent in the nature of the study.
In summary, targeting synovial membrane, synovial fluid, articular cartilage, and subchondral bone with intra-articular injections and intraosseous infiltrations of PRP reduces pain and MSCs in SF, besides significantly improving knee joint function in patients with severe knee OA, with no adverse event reported. This work aims to be a first step for further research in this field, both in basic research and in increasingly robust clinical trials.
This trial is approved by Clinical Research Ethics Committee of the Basque Country.
Sabino Padilla is scientist at BTI Biotechnology Institute, a dental implant company that investigates the fields of oral implantology and PRGF-Endoret technology. The other authors have no potential competing interests.
The authors wish to thank M. Sánchez, A. Iriondo, M. Montoya, M. M. González, and I. Rodrigo for their efficient data collection.