- Email: [email protected]
Injecting autologous platelet rich plasma solely into the knee joint is not adequate in treating geriatric patients with moderate to severe knee osteoarthritis
Experimental Gerontology 119 (2019) 1–6
Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Injecting autologous platelet rich plasma solely into the knee joint is not adequate in treating geriatric patients with moderate to severe knee osteoarthritis
T
⁎
Carl P.C. Chena, , Jean-Lon Chena, Chih-Chin Hsub, Yu-Cheng Peia, Wei-Han Changa, Hsueh-Chih Lua a Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan City, Taiwan b Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Keelung and College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan City, Taiwan
A R T I C LE I N FO
A B S T R A C T
Section Editor: Christiaan Leeuwenburgh
Knee pain caused by osteoarthritis (OA) is commonly seen in geriatric patients. Patients with knee OA are often complicated with joint pain, soreness, and weakness. The injection of autologous platelet rich plasma (aPRP) has been proven to be effective in treating mild knee OA. The effect of injecting aPRP in treating moderate to severe degrees of knee OA remains controversial. This study aimed to evaluate the effectiveness of aPRP in treating patients with at least grade 2 on the Kellgren and Lawrence system for the classification of knee OA using a proteomic approach and clinical evaluation tool of Lequesne index. Musculoskeletal ultrasound was used for accurate needle placement into the knee joint, and to the perimeniscal soft tissue for the injection of aPRP. Three monthly aPRP injections were performed. Group 1 patients received intra-articular (IA) injection only, while group 2 received simultaneous IA and pes anserinus aPRP injections. After two monthly aPRP injections, both groups revealed significant drops in average SF total protein concentrations, and increases in the protein concentrations associated with chelation and anti-aging (eg/transthyretin, matrilin, and complement). However, it is group 2 that revealed significant decreases in the protein concentrations associated with inflammation (eg/ immunoglobulin and apolipoprotein), and improved knee functional status. SF appeared to become less susceptible to degeneration after aPRP injections in group 2. As a result, at least 2 monthly injection of IA aPRP in conjunction with accurate injection of aPRP to the perimeniscal soft tissue structure such as the pes anserinus may be a viable option in treating patients with moderate to severe degrees of knee OA.
1. Introduction Knee osteoarthritis (OA) is a common cause of lower limb pain in the aging population. Elderly patients with knee osteoarthritis (OA) are often complicated with joint soreness, weakness, swelling, and pain (Kon et al., 2011). Excessive amount of synovial fluid (SF) accumulated in the bursae around the knee joint is the most frequently observed cause of these symptoms. Out of all the bursae surrounding the knee joint, supra-patellar bursitis is most often associated with knee pain (de Miguel Mendieta et al., 2006). Musculoskeletal ultrasound is now the preferred imaging tool in diagnosing bursitis as it is radiation free (Lueders et al., 2016). In patients with moderate to severe degrees of knee OA, malalignment of the knee joint frequently occurs. This may cause excess ⁎
stress onto the pes anserinus, medial collateral ligament (MCL), and inducing medial knee space narrowing causing the medial meniscus to be protruded. As a result, pes anserine bursitis, tendinitis, MCL sprain and meniscus injuries the likely causes of generating knee pain (Uysal et al., 2015). More than 80% of patients with pes anserinus bursitis or tendinitis are associated with knee OA (Yoon et al., 2005). Treatments of knee OA include taking oral nonsteroidal anti-inflammatory drugs (NSAIDs), physical modalities, strengthening exercise, shoe modification, and oral supplements such as glucosamine (Wang et al., 2016). The concept of viscosupplementation has been widely applied in the treatment of knee OA. It is based on the replacement of SF using hyaluronic acid (HA) (Cohen et al., 2008). But in recent years, a more regenerative concept of using blood derivative of autologous platelet-rich plasma (aPRP) has been used in treating knee OA. Studies have shown
Corresponding author at: Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Linkou, Taiwan. E-mail addresses: [email protected], [email protected] (C.P.C. Chen).
https://doi.org/10.1016/j.exger.2019.01.018 Received 13 August 2018; Received in revised form 25 December 2018; Accepted 15 January 2019 Available online 19 January 2019 0531-5565/ © 2019 Published by Elsevier Inc.
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
Fig. 1. Musculoskeletal ultrasound image of the perimeniscal soft tissue structures of the medial knee joint. The injection needle is clearly shown. The distance between “*” is the medial knee joint.
this study. They signed the informed consent before participating in this study. This study was conducted in a tertiary hospital rehabilitation outpatient clinic, and was approved by the local medical ethics and the human clinical trial committee (IRB: 201600291A3 & 104-9766A3). The inclusion criteria were:
that the effect of aPRP in treating knee OA is superior than that of HA, and should be applied to significantly improve pain and functional scales in early stages of knee OA (Laudy et al., 2015). Whether aPRP can be beneficial to patients with moderate to severe degrees of knee OA remains controversial. The biological rational behind aPRP treatment is that pools of growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β) are stored in platelet α-granules. These granules take part in the repair and regeneration of articular cartilage, tendons, and ligaments (Khoshbin et al., 2013). However, when treating patients with severe degrees of knee OA, injecting aPRP solely into the knee joint may not be enough. Since the biomechanical changes that occur after knee joint mal-alignment, pes anserine bursitis, MCL sprain and meniscus injuries are also the likely causes of knee pain and antalgic gait pattern in these patients. As a result, aPRP injection to these perimeniscal soft tissue structures may be needed as well. This study is to examine whether the combination of injecting aPRP injection into the knee joint and to the perimeniscal MCL/pes anserinus areas is superior to that of injecting aPRP solely into the knee joint. Proteomics and functional pain scale evaluations will be used to judge the treatment effectiveness. The Bradford method will be used to calculate the SF total protein concentrations, and the 2-dimensional electrophoresis (2-DE) proteomic technique will be used to calculate the changes in individual SF protein concentrations before and after aPRP injections (Olson, 2016). The 2-DE technique is capable of detecting hundreds of protein spots simultaneously on gel images (Gorg et al., 2004). We hypothesize that simultaneous injection of aPRP into the knee joint and to the MCL/pes anserinus sites are more effective in treating patients with moderate to severe degrees of knee OA. Autologous PRP injection to the pes anserinus may strengthen the pes anserine tendons and alleviate the bursitis condition. Injecting aPRP to the MCL to cover the medial meniscus can ensure the repair and regeneration to these soft tissue structures, enabling the meniscus to be more robust, which can result in less pain during walking (Bagwell et al., 2018). Results obtained in this study may help us in finding a suitable aPRP injection approach that can be applied in treating patients with moderate to severe degrees of knee OA.
1. Thickness of the supra-patellar bursa was measured to be > 2 mm (mm) as confirmed by musculoskeletal ultrasound. 2. Patients had history of receiving knee joint steroid injections, physical modality treatments, and taking oral NSAIDs but without any obvious reduction in SF volumes and knee pain. 3. Protruded medial meniscus causing MCL sprain as confirmed by musculoskeletal ultrasound. 4. Increased hypoechoic changes at the pes anserinus complex, indicating inflammation of the tendons and bursitis, as confirmed by musculoskeletal ultrasound. 5. The major cause of the supra-patellar bursitis, protruded meniscus with bulging MCL, and pes anserine inflammation was due to knee OA, not caused by infectious or inflammatory knee disorders. 6. Ultrasound confirmed that the supra-patellar bursa was in communication with the synovial cavity of the knee joint. This was to rule out the diagnosis of isolated cystic lesion at the supra-patellar region. 2.1. Treatment protocol The “REGEN” environmental chamber for storage of platelet concentrate kit (REGENLAB, RegenACR-C Classic, Switzerland) was used for the preparation of aPRP. After harvesting 10 mL of blood from the patient, it was then injected into the PRP test tube. Centrifugation was then performed for 15 min under the speed of 3400 rounds per minute (rpm). Approximately 6 mL of supernatant (PRP) was then aspirated from the test tube and was ready to be used for injection. The recruited patients were divided into 2 groups. Group 1 (n = 15) received 4 mL of aPRP injection to the knee joint only. Groups 2 (n = 15) received 4 mL of aPRP injection into the knee joint, and 2 mL of aPRP injection to the pes anserinus complex. Adjustment of needle depth and bevel may be needed in order for aPRP to effectively bath the pes anserine bursa, tendons (gracilis, sartorius, and semitendinosus), MCL, and the medial meniscus (Fig. 1). Musculoskeletal ultrasound was used to accurately guide the needle into the supra-patellar bursa site for the aspiration of SF, intra-articular injection of aPRP, and also injection of aPRP to the pes anserinus
2. Materials and methods In this study, a total of 30 elderly patients diagnosed with at least grade 2 on the Kellgren and Lawrence system for classification of osteoarthritis of knee and with supra-patellar bursitis were recruited into 2
Experimental Gerontology 119 (2019) 1–6
7.84 ± 2.84a,b
9.85 ± 2.78
complex. Injection was performed on a once per month basis for a total of three months. The Tablet Ultrasound Imaging System T3300 ultrasound system with a bandwidth of 4–15 MHz linear transducer was used (BenQ Medical Technology Corporation, Taipei). Accurate placement of the needle into the bursa can avoid poking into the muscle or other soft tissues that may cause blood contamination in the SF. The standard lateral approach with the knee extended was applied for ultrasound-guided SF aspiration and IA injection. The interpretation of ultrasound images and the injection procedures were done by the same physician to avoid experimental bias.
6.46 ± 2.66 7.11 ± 2.47
a,b
The proteomic technique of 2-DE was used to detect significant increases and decreases in protein spot intensities before and after PRP injections. Steps of 2-DE and subsequent protein identification using mass spectrometry (MS) can be read in our previous published article (Chen et al., 2014). SYPRO Ruby stained SF 2-DE gel images were obtained from each patient. Triplicate gel images were constructed for each patient's SF sample gathered before the 1st, 2nd, 3rd aPRP injections, and 1, 3, and 6 months after the completion of aPRP injections. The ProXPRESS™ 2D Proteomic Image System (PerkinElmer Life and Analytical Sciences) was used to scan the SYPRO Ruby stained SF 2-DE gels. The gel images were acquired as digital TIF files and analyzed using the PDQuest Basic 8.0.1 Analytical software (BioRad).
Values expressed as mean ± standard deviation (SD). a Statistical comparison between the time of treatment (or time of follow up after the completion of all 3 PRP injections) and the time of 1st PRP injection (p < 0.01). b Statistical comparison between the time of treatment (or time of follow up after the completion of all 3 PRP injections) and the time of 2nd PRP injection (p < 0.01).
13.92 ± 2.51 14.71 ± 3.44
6.88 ± 2.11
a,b
8.97 ± 2.59 10.02 ± 2.48 9.78 ± 2.61 12.76 ± 2.89 13.97 ± 2.96
a,b
23.75 ± 3.14a,b 24.97 ± 3.88a,b 32.62 ± 3.71 34.49 ± 4.07
25.54 ± 2.74a,b
27.14 ± 3.11a,b 25.72 ± 3.13a,b 33.57 ± 2.58 35.38 ± 3.78
Average SF Total Protein Concentration In μg/μL (Group 1) Average SF Total Protein Concentration In μg/μL (Group 2) Lequesne Functional Index (Group 1) Lequesne Functional Index (Group 2)
Measured parameters
1 month after the completion of all 3 monthly PRP injections Time of 3rd PRP injection (1 month after the 2nd PRP injection) Time of 2nd PRP injection (1 month after the 1st PRP injection) Time of 1st PRP injection
26.47 ± 2.23a,b
3 months after the completion of all 3 monthly PRP injections
2.2. 2-Dimension electrophoresis (2-DE)
Experimental time periods
Table 1 Differences in synovial fluid protein concentrations, and LeQuesne Index before and after aPRP injections.
26.28 ± 3.69a
25.86 ± 3.88a,b
6 months after the completion of all 3 monthly PRP injections
C.P.C. Chen et al.
2.3. Western immunoblotting Western immunoblotting method was used to validate the protein spots that revealed significant increases or decreases in concentrations after 2-DE analyses. Polyclonal and monoclonal antibodies were purchased from the ABCAM company (ABCAM, Cambridge, MA, USA) to detect the proteins of apolipoprotein A-I (APOA1), complement 5 (C5), haptoglobin (HPT), immunoglobulin kappa chain (IGKC), matrilin, MATNtransferrin (TRFE), transthyretin (TTR), and matrix metalloproteinase (MMP). Experimental steps of Western immunoblotting can be seen in our previous published article (Chen et al., 2014). The protein band intensities were analyzed using the ChemiCapture Software. 2.4. Calculation of synovial fluid total protein concentrations The standard curve of known bovine serum albumin (BSA) (Sigma, > 96% purity) concentrations (2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg) was constructed to calculate the total protein concentration of SF samples. The Bradford method was applied for the calculation of protein concentrations (Olson, 2016). 2.5. Lequesne Index The Lequesne Functional Index for degenerative knee joint was used to evaluate the extent of knee pain, and the changes of knee functional status before and after PRP injections. The Lequesne Functional Index was evaluated by the same experimenter to prevent inter-tester variability. Index value > 7 is highly indicative of degenerative knee disorder (da Silva et al., 2015). 2.6. Statistical analysis The Wilcoxon signed-rank test was used to compare the age difference between patients. The statistical tool of repeated measures ANOVA with Bonferroni correction was used to compare the SF total protein and individual protein concentrations, and changes in Lequesne Functional Index values before and after aPRP injections. Protein concentrations, and functional index values were calculated and measured at the time periods of the 1st, 2nd, 3rd aPRP injections. The follow up time periods were 1, 3 and 6 months after the completion of all 3 aPRP 3
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
validations were performed before the time of the first PRP injection and one month after the completion all 3 monthly PRP injections.
injections. The Statistical Program for Social Sciences (SPSS) program (SPSS Inc., Chicago) was used for data calculations. Values with p < 0.01 were considered statistically significant.
4. Discussion 3. Results In most of the studies related to knee OA injection treatment using aPRP, injection is done mainly into the knee joints only. Simultaneous aPRP injection into the knee joint and to the perimeniscal soft tissue structures such as the MCL, medial meniscus, and pes anserinus are seldom reported. Studies related to aPRP injection to the perimeniscal soft tissue structure such as MCL are mostly related to sport injuries or traumatic events (Bagwell et al., 2018). The efficacy of aPRP in treating patients with moderate to severe degrees of knee OA remains controversial. Although aPRP has been shown to be effective in treating patients with early and mild knee OA, it is recommended that at least 2 monthly injections are needed in order for the effect of aPRP to be more robust (Chen et al., 2017). Musculoskeletal ultrasound can be used as a tool in accurately guiding the needle to the target site that is to be treated (Chen et al., 2017). The injection needle can be accurately guided to the pes anserine bursa, tendons, MCL and the medial meniscus, and administer aPRP solution to these sites. It is technically feasible to inject to these areas under one injection spot. In other words, multiple insertion and retraction needle motions on several injection spots can be avoided using the ultrasound guidance. For instance, needle can be guided to the pes anserine tendons, then by adjusting the bevel, the needle can also be placed at the MCL for further aPRP injections, all under one needle insertion site. SF total protein concentrations in both groups revealed significant decreases after the second monthly PRP injections. Group 2 was associated with at least two-fold concentration decreases in the proteins of APOA1, MMP, IGKC, HPT, and TRFE. In SF, abundant proteins of albumin and immunoglobulin constitute about 60 to 80% of the total protein concentration (Chen et al., 2014). Therefore, significant decrease in the protein concentration of immunoglobulin may be the main cause of decreased SF total protein concentration after aPRP injections. Although the extent of inflammation in the synovial membrane of knee OA is not as obvious as in rheumatoid arthritis (RA), it is a likely ongoing process in knee OA as cartilage destruction can be observed (Chang et al., 2009). APOA1 is a plasma derived high density apolipoprotein that plays a key role in the transport of lipids. Dysregulated lipid profile in the SF is an underlying cause of inflammation in the knee joint cavity and
The mean age of the 30 recruited patients (> 65 years of age, 18 female and 12 male patients) was 73 ± 3.4 years. Wilcoxon signedrank test revealed no significant statistical differences in their age. In group 1, the average SF total protein concentrations were calculated to be 35.38 ± 3.78 μg/μL (group 2 = 34.49 ± 4.07 μg/μL) at the time of 1st aPRP injection, 33.57 ± 2.58 μg/μL (group 2 = 32.62 ± 3.71 μg/ μL) at the time of 2nd aPRP injection, 25.72 ± 3.13 μg/μL (group 2 = 24.97 ± 3.88 μg/μL) at the time of 3rd aPRP injection, and 27.14 ± 3.11 μg/μL (group 2 = 23.75 ± 3.14 μg/μL) at 1 month, 26.47 ± 2.23 μg/μL (group 2 = 25.54 ± 2.74 μg/μL) at 3 months and 25.86 ± 3.88 μg/μL (group 2 = 26.28 ± 3.69 μg/μL) at 6 months after the completion of all 3 aPRP injections. Significant decreases in SF total protein concentrations were observed after receiving 2 PRP injections in both groups (p < 0.01) (Table 1). There were no significant changes in SF total protein concentrations in both groups after receiving only the 1st PRP injection. In terms of average Lequesne Functional Index, group 1 was calculated to be 13.97 ± 2.96 (group 2 = 14.71 ± 3.44) at the time of 1st aPRP injection, 12.76 ± 2.89 (group 2 = 13.92 ± 2.51) at the time of 2nd aPRP injection, 9.78 ± 2.61 (group 2 = 7.11 ± 2.47) at the time of 3rd aPRP injection, and 10.02 ± 2.48 (group 2 = 6.46 ± 2.66) at 1 month, 8.97 ± 2.59 (group 2 = 6.88 ± 2.11) at 3 months, and 9.85 ± 2.78 (group 2 = 7.84 ± 2.84) at 6 months after the 3rd PRP injection. Significant decreases in Lequesne Functional Index were observed at the time of the 3rd PRP injection (after the completion of 2 monthly PRP injections), and in all the follow up time periods (p < 0.01) (Table 1). The 2-DE gel images of SF samples revealed at least 2-fold increases in protein intensities of matrilin, transthyretin, and complement 5 in both groups. Group 2 revealed significant decreases in the protein concentrations of haptoglobin, immunoglobulin kappa chain, transferrin, apolipoprotein A-I, and matrix metalloproteinase for at least 2fold. Decreases in the concentrations of these proteins were not significant in group 1. These protein intensities were further validated by Western immunoblotting (Table 2 states the names of SF proteins confirmed by MS and Fig. 2 reveals the images of proteins that were identified by 2-DE). Western immunoblotting protein intensity
Table 2 Protein names confirmed by mass spectrometry after 2 – DE separation and concentration differences validated by Western immunoblotting. Spot namea
Description
Access Noa
Mr (kDa)b
pIb
No. of matchedc
Seq cov (%)d
TTR MATN C5 APOA1 MMP IGKC HPT TRFE
Transthyretin ↑+ Matrilin ↑+ Complement 5 ↑+ Apolipoprotein A-1 ↓⁎ Matrix metalloproteinase ↓⁎ Immunoglobulin kappa chain ↓⁎ Haptoglobin ↓⁎ Transferrin ↓⁎
P02766 P21941 P06684 P02647 P14780 P01834 P00738 P02787
15.87 54.00 112.75 30.00 92.00 11.61 45.80 79.29
5.52 8.31 8.90 5.56 5.70 9.10 6.13 6.81
10 36 18 13 17 47 20 24
73 51 67 46 38 66 44 36
↑: increased in concentration. ↓: decreased in concentration. a Protein name and accession number according to the SwissProt and TrEMBL databases. b Predicted molecular weights in kiloDaltons and pI according to protein sequence and Swiss-2DPAGE databases. c Number of peptide masses matching the top hit from MS-Fit PMF. d Aminoacidic sequence coverage for the identified proteins. + Western immunoblotting protein spot intensity validation comparison conducted between the time of first PRP injection and one month after the completion of all 3 monthly PRP injections in both groups (p < 0.01). ⁎ Western immunoblotting protein spot intensity validation comparison conducted between the time of first PRP injection and one month after the completion of all 3 monthly PRP injections in group 2 (p < 0.01). 4
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
Fig. 2. SYPRO Ruby stained 2-DE gel image of the SF sample. The protein spot names and their molecular weights (MW) in kiloDaltons (kDa) were confirmed by mass spectrometry.
increased chondrocyte hypertrophy is observed, which is associated with cartilage degeneration. As a result, increased matrilin concentration after PRP injections may inhibit chondrocyte hypertrophy, and further attenuates cartilage degeneration (Yang et al., 2014). The complement system complements the ability of antibodies and phagocytic cells to clear pathogens. C5 is associated with degenerative joint disorders as its activation is abnormally high in human OA joints (Wang et al., 2011). Our study revealed similar findings. Therefore, increased C5 concentration may suggest improved clearance of pathogens associated with knee OA. APOA1, MMP, IGKC, HPT, and TRFE revealed significant decreases in concentrations in group 2 only. A logical way to explain this phenomenon would be that in patients with moderate to severe degrees of knee OA, joint space narrowing and perimeniscal soft tissue inflammation exist. Although the rheology of SF has changed after aPRP injections (ex/protein concentration associated with inflammation decreased and protein concentration associated with chelation increased), friction of the narrowed knee joint, and inflammation of the perimeniscal soft tissue such as the pes anserine tendons and MCL are constantly occurring. This may be the reason why group 1 patients still experience knee pain after numerous IA aPRP injections, and the decrease in the concentrations of these proteins did not reach statistical significance. After the perimeniscal soft tissue structures are injected with aPRP, the injured MCL can be healed, and the degree of inflammation at pes anserine tendons and bursa can be decreased, ensuring less laxity of these structures (Mastrangelo et al., 2011). This explains why knee pain and functional status are significantly improved when aPRP are injected both into the knee joint and to the perimeniscal soft tissue structures. In summary, results in this study revealed that the injection of aPRP can be beneficial for patients with moderate to severe degrees of knee OA. But it is strongly recommended that aPRP should be simultaneously injected into the knee joint, and also to the perimeniscal pes anserinus. Then with the adjustment of needle depth and bevel, aPRP can bath the soft tissue structures of pes anserine tendons, bursa, MCL, and medial meniscus. Multiple monthly injections are recommended in order for
induces the development of OA. The pro-inflammatory property of APOA1 was confirmed in an in vitro environment and closely associated with the protein matrix metalloproteinases (MMPs). APOA1 plays an important role in inducing strong MMP expressions (Oliviero et al., 2009). MMPs can aggravate inflammation in OA by degrading extracellular matrix macromolecules and decreasing expression of chondrocyte proteins, resulting in severe joint pain, loss of movement, and irreversible dysfunction (Lu et al., 2015). As a result, decreases in APOA1 and MMP concentrations after aPRP injections may attenuate knee joint inflammatory responses. IGKC is one of the two types of light chains that is involved in antibody production as secreted by B lymphocytes. Increased levels of free Ig light chains are detected in inflammation (Chang et al., 2009). HPT is a major acute phase glycoprotein with a molecular weight of 43 kDa. When the concentration of HPT increases by twofold or more in the SF, it is correlated with conditions such as inflammation, injury or malignancy (Park et al., 2013). TRFE is an iron-binding plasma glycoprotein that is responsible with the control of free iron in biological fluids. A study has shown that transferrin, immunoglobulin, and albumin protein concentrations are increased in OA and RA joints (Izai et al., 1992). Therefore, significant decreases in IGKC, HPT, and TRFE proteins suggest that the extent of knee joint inflammation in OA patients is decreased after aPRP injections. Three SF proteins revealed significant increases in concentrations for > 2-folds in both groups after the completion of aPRP injections. The deposition of amyloid is a risk factor in the development of OA, and increased amyloid concentration can be observed in OA knee joints. TTR protein can chelate amyloid and further preventing the deposition of amyloid in knee joints. TTR is a homotetrameric protein synthesized mainly in the choroid plexus and liver, and can be detected predominantly at the cartilage surface of aged normal cartilage samples (Akasaki et al., 2015). Therefore, significant increases in SF TTR concentrations after aPRP injections may signify a healthier knee joint as more amyloid deposits can be chelated. Matrilin protein is an extracellular matrix protein that is believed to play a crucial regulatory role in maintaining cartilage microenvironment. In matrilin knock-out mice, 5
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
the treatment of aPRP to be effective. Results in this study showed that at least two monthly aPRP injections are needed in order to achieve significant proteomic changes, and improvements in pain and functional scales. In future related research, our goal will be to conduct studies with larger sample sizes, hoping that more SF protein biomarkers related to knee OA can be discovered. Other simultaneous injection techniques will also be studied and applied, such as injecting aPRP to the genicular nerves, and to observe whether the effect of aPRP can be potentiated in patients with moderate to severe degrees of knee OA.
Chen, C.P., Hsu, C.C., Pei, Y.C., Chen, R.L., Zhou, S., Shen, H.C., Lin, S.C., Tsai, W.C., 2014. Changes of synovial fluid protein concentrations in supra-patellar bursitis patients after the injection of different molecular weights of hyaluronic acid. Exp. Gerontol. 52, 30–35. Chen, C.P.C., Cheng, C.H., Hsu, C.C., Lin, H.C., Tsai, Y.R., Chen, J.L., 2017. The influence of platelet rich plasma on synovial fluid volumes, protein concentrations, and severity of pain in patients with knee osteoarthritis. Exp. Gerontol. 93, 68–72. Cohen, M.M., Altman, R.D., Hollstrom, R., Hollstrom, C., Sun, C., Gipson, B., 2008. Safety and efficacy of intra-articular sodium hyaluronate (Hyalgan) in a randomized, double-blind study for osteoarthritis of the ankle. Foot Ankle Int. 29, 657–663. da Silva, F.S., de Melo, F.E., do Amaral, M.M., Caldas, V.V., Pinheiro, I.L., Abreu, B.J., Vieira, W.H., 2015. Efficacy of simple integrated group rehabilitation program for patients with knee osteoarthritis: single-blind randomized controlled trial. J. Rehabil. Res. Dev. 52, 309–322. de Miguel Mendieta, E., Cobo Ibanez, T., Uson Jaeger, J., Bonilla Hernan, G., Martin Mola, E., 2006. Clinical and ultrasonographic findings related to knee pain in osteoarthritis. Osteoarthr. Cartil. 14, 540–544. Gorg, A., Weiss, W., Dunn, M.J., 2004. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685. Izai, M., Miyazaki, S., Murai, R., Morioka, Y., Hayashi, H., Nishiura, M., Miura, K., 1992. Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis. Endocrinol. Jpn. 39, 259–267. Khoshbin, A., Leroux, T., Wasserstein, D., Marks, P., Theodoropoulos, J., Ogilvie-Harris, D., Gandhi, R., Takhar, K., Lum, G., Chahal, J., 2013. The efficacy of platelet-rich plasma in the treatment of symptomatic knee osteoarthritis: a systematic review with quantitative synthesis. Arthroscopy 29, 2037–2048. Kon, E., Mandelbaum, B., Buda, R., Filardo, G., Delcogliano, M., Timoncini, A., Fornasari, P.M., Giannini, S., Marcacci, M., 2011. Platelet-rich plasma intra-articular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy 27, 1490–1501. Laudy, A.B., Bakker, E.W., Rekers, M., Moen, M.H., 2015. Efficacy of platelet-rich plasma injections in osteoarthritis of the knee: a systematic review and meta-analysis. Br. J. Sports Med. 49, 657–672. Lu, S., Xiao, X., Cheng, M., 2015. Matrine inhibits IL-1beta-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-kappaB in human chondrocytes in vitro. Int. J. Clin. Exp. Pathol. 8, 4764–4772. Lueders, D.R., Smith, J., Sellon, J.L., 2016. Ultrasound-guided knee procedures. Phys. Med. Rehabil. Clin. N. Am. 27, 631–648. Mastrangelo, A.N., Vavken, P., Fleming, B.C., Harrison, S.L., Murray, M.M., 2011. Reduced platelet concentration does not harm PRP effectiveness for ACL repair in a porcine in vivo model. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 29, 1002–1007. Oliviero, F., Sfriso, P., Baldo, G., Dayer, J.M., Giunco, S., Scanu, A., Bernardi, D., Ramonda, R., Plebani, M., Punzi, L., 2009. Apolipoprotein A-I and cholesterol in synovial fluid of patients with rheumatoid arthritis, psoriatic arthritis and osteoarthritis. Clin. Exp. Rheumatol. 27, 79–83. Olson, B.J., 2016. Assays for determination of protein concentration. Curr. Protoc. Pharmacol. 73 A.3a.1-a.3a.32. Park, H.J., Oh, M.K., Kim, N.H., Cho, M.L., Kim, I.S., 2013. Identification of a specific haptoglobin C-terminal fragment in arthritic synovial fluid and its effect on interleukin-6 expression. Immunology 140, 133–141. Uysal, F., Akbal, A., Gokmen, F., Adam, G., Resorlu, M., 2015. Prevalence of pes anserine bursitis in symptomatic osteoarthritis patients: an ultrasonographic prospective study. Clin. Rheumatol. 34, 529–533. Wang, P., Yang, L., Li, H., Lei, Z., Yang, X., Liu, C., Jiang, H., Zhang, L., Zhou, Z., Reinhardt, J.D., He, C., 2016. Effects of whole-body vibration training with quadriceps strengthening exercise on functioning and gait parameters in patients with medial compartment knee osteoarthritis: a randomised controlled preliminary study. Physiotherapy 102, 86–92. Wang, Q., Rozelle, A.L., Lepus, C.M., Scanzello, C.R., Song, J.J., Larsen, D.M., Crish, J.F., Bebek, G., Ritter, S.Y., Lindstrom, T.M., Hwang, I., Wong, H.H., Punzi, L., Encarnacion, A., Shamloo, M., Goodman, S.B., Wyss-Coray, T., Goldring, S.R., Banda, N.K., Thurman, J.M., Gobezie, R., Crow, M.K., Holers, V.M., Lee, D.M., Robinson, W.H., 2011. Identification of a central role for complement in osteoarthritis. Nat. Med. 17, 1674–1679. Yang, X., Trehan, S.K., Guan, Y., Sun, C., Moore, D.C., Jayasuriya, C.T., Chen, Q., 2014. Matrilin-3 inhibits chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist. J. Biol. Chem. 289, 34768–34779. Yoon, H.S., Kim, S.E., Suh, Y.R., Seo, Y.I., Kim, H.A., 2005. Correlation between ultrasonographic findings and the response to corticosteroid injection in pes anserinus tendinobursitis syndrome in knee osteoarthritis patients. J. Korean Med. Sci. 20, 109–112.
5. Conclusion This study aimed to examine whether the injection of aPRP is effective for patients with moderate to severe degrees of knee OA. Results in this study showed that in the group of patients who received simultaneous aPRP injection intra-articularly and to the perimeniscal soft tissue of pes anserine, pain and functional scores were significantly improved. The concentrations of SF total protein, and proteins associated with inflammation (eg/APOA1, MMP, IGKC, HPT, and TRFE) were significantly decreased. Protein concentrations associated with chelation and anti-aging physiological functions (eg/TTR, MATN, and C5) were increased significantly. As a result, the rheology of knee SF appears to become less susceptible to degeneration after aPRP injections. As a result, at least 2 monthly injection of IA aPRP in conjunction with accurate injection of aPRP to the perimeniscal soft tissue structure such as the pes anserinus may be a viable option in treating patients with moderate to severe degrees of knee OA. Conflict of interest All the authors in this study have no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgements This study was supported by the grants from the National Science Council, Taiwan (NMRPG3F6261-2 (NMRPD1F1642), 105-2314-B182A-023-MY2) and the Chang Gung Memorial Hospital at Linkou Research Project Grants of CMRPG1F0091-2 to Dr. Carl P.C. Chen. The National Science Council grant supported the expenses of consumable products. The Chang Gung Memorial Hospital Research Project Grant supported the cost of western immunoblotting analyses and the purchase of antibodies. References Akasaki, Y., Reixach, N., Matsuzaki, T., Alvarez-Garcia, O., Olmer, M., Iwamoto, Y., Buxbaum, J.N., Lotz, M.K., 2015. Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis. Arthritis Rheum. 67, 2097–2107. Bagwell, M.S., Wilk, K.E., Colberg, R.E., Dugas, J.R., 2018. The use of serial platelet RICH plasma injections with early rehabilitation to expedite grade iii medial collateral ligament injury in a professional athlete: a CASE report. Int. J. Sports Phys. Ther. 13, 520–525. Chang, X., Cui, Y., Zong, M., Zhao, Y., Yan, X., Chen, Y., Han, J., 2009. Identification of proteins with increased expression in rheumatoid arthritis synovial tissues. J. Rheumatol. 36, 872–880.
6
Contents lists available at ScienceDirect
Experimental Gerontology journal homepage: www.elsevier.com/locate/expgero
Injecting autologous platelet rich plasma solely into the knee joint is not adequate in treating geriatric patients with moderate to severe knee osteoarthritis
T
⁎
Carl P.C. Chena, , Jean-Lon Chena, Chih-Chin Hsub, Yu-Cheng Peia, Wei-Han Changa, Hsueh-Chih Lua a Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Linkou and College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan City, Taiwan b Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Keelung and College of Medicine, Chang Gung University, Kwei-Shan, Tao-Yuan City, Taiwan
A R T I C LE I N FO
A B S T R A C T
Section Editor: Christiaan Leeuwenburgh
Knee pain caused by osteoarthritis (OA) is commonly seen in geriatric patients. Patients with knee OA are often complicated with joint pain, soreness, and weakness. The injection of autologous platelet rich plasma (aPRP) has been proven to be effective in treating mild knee OA. The effect of injecting aPRP in treating moderate to severe degrees of knee OA remains controversial. This study aimed to evaluate the effectiveness of aPRP in treating patients with at least grade 2 on the Kellgren and Lawrence system for the classification of knee OA using a proteomic approach and clinical evaluation tool of Lequesne index. Musculoskeletal ultrasound was used for accurate needle placement into the knee joint, and to the perimeniscal soft tissue for the injection of aPRP. Three monthly aPRP injections were performed. Group 1 patients received intra-articular (IA) injection only, while group 2 received simultaneous IA and pes anserinus aPRP injections. After two monthly aPRP injections, both groups revealed significant drops in average SF total protein concentrations, and increases in the protein concentrations associated with chelation and anti-aging (eg/transthyretin, matrilin, and complement). However, it is group 2 that revealed significant decreases in the protein concentrations associated with inflammation (eg/ immunoglobulin and apolipoprotein), and improved knee functional status. SF appeared to become less susceptible to degeneration after aPRP injections in group 2. As a result, at least 2 monthly injection of IA aPRP in conjunction with accurate injection of aPRP to the perimeniscal soft tissue structure such as the pes anserinus may be a viable option in treating patients with moderate to severe degrees of knee OA.
1. Introduction Knee osteoarthritis (OA) is a common cause of lower limb pain in the aging population. Elderly patients with knee osteoarthritis (OA) are often complicated with joint soreness, weakness, swelling, and pain (Kon et al., 2011). Excessive amount of synovial fluid (SF) accumulated in the bursae around the knee joint is the most frequently observed cause of these symptoms. Out of all the bursae surrounding the knee joint, supra-patellar bursitis is most often associated with knee pain (de Miguel Mendieta et al., 2006). Musculoskeletal ultrasound is now the preferred imaging tool in diagnosing bursitis as it is radiation free (Lueders et al., 2016). In patients with moderate to severe degrees of knee OA, malalignment of the knee joint frequently occurs. This may cause excess ⁎
stress onto the pes anserinus, medial collateral ligament (MCL), and inducing medial knee space narrowing causing the medial meniscus to be protruded. As a result, pes anserine bursitis, tendinitis, MCL sprain and meniscus injuries the likely causes of generating knee pain (Uysal et al., 2015). More than 80% of patients with pes anserinus bursitis or tendinitis are associated with knee OA (Yoon et al., 2005). Treatments of knee OA include taking oral nonsteroidal anti-inflammatory drugs (NSAIDs), physical modalities, strengthening exercise, shoe modification, and oral supplements such as glucosamine (Wang et al., 2016). The concept of viscosupplementation has been widely applied in the treatment of knee OA. It is based on the replacement of SF using hyaluronic acid (HA) (Cohen et al., 2008). But in recent years, a more regenerative concept of using blood derivative of autologous platelet-rich plasma (aPRP) has been used in treating knee OA. Studies have shown
Corresponding author at: Department of Physical Medicine & Rehabilitation, Chang Gung Memorial Hospital at Linkou, Taiwan. E-mail addresses: [email protected], [email protected] (C.P.C. Chen).
https://doi.org/10.1016/j.exger.2019.01.018 Received 13 August 2018; Received in revised form 25 December 2018; Accepted 15 January 2019 Available online 19 January 2019 0531-5565/ © 2019 Published by Elsevier Inc.
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
Fig. 1. Musculoskeletal ultrasound image of the perimeniscal soft tissue structures of the medial knee joint. The injection needle is clearly shown. The distance between “*” is the medial knee joint.
this study. They signed the informed consent before participating in this study. This study was conducted in a tertiary hospital rehabilitation outpatient clinic, and was approved by the local medical ethics and the human clinical trial committee (IRB: 201600291A3 & 104-9766A3). The inclusion criteria were:
that the effect of aPRP in treating knee OA is superior than that of HA, and should be applied to significantly improve pain and functional scales in early stages of knee OA (Laudy et al., 2015). Whether aPRP can be beneficial to patients with moderate to severe degrees of knee OA remains controversial. The biological rational behind aPRP treatment is that pools of growth factors such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF-β) are stored in platelet α-granules. These granules take part in the repair and regeneration of articular cartilage, tendons, and ligaments (Khoshbin et al., 2013). However, when treating patients with severe degrees of knee OA, injecting aPRP solely into the knee joint may not be enough. Since the biomechanical changes that occur after knee joint mal-alignment, pes anserine bursitis, MCL sprain and meniscus injuries are also the likely causes of knee pain and antalgic gait pattern in these patients. As a result, aPRP injection to these perimeniscal soft tissue structures may be needed as well. This study is to examine whether the combination of injecting aPRP injection into the knee joint and to the perimeniscal MCL/pes anserinus areas is superior to that of injecting aPRP solely into the knee joint. Proteomics and functional pain scale evaluations will be used to judge the treatment effectiveness. The Bradford method will be used to calculate the SF total protein concentrations, and the 2-dimensional electrophoresis (2-DE) proteomic technique will be used to calculate the changes in individual SF protein concentrations before and after aPRP injections (Olson, 2016). The 2-DE technique is capable of detecting hundreds of protein spots simultaneously on gel images (Gorg et al., 2004). We hypothesize that simultaneous injection of aPRP into the knee joint and to the MCL/pes anserinus sites are more effective in treating patients with moderate to severe degrees of knee OA. Autologous PRP injection to the pes anserinus may strengthen the pes anserine tendons and alleviate the bursitis condition. Injecting aPRP to the MCL to cover the medial meniscus can ensure the repair and regeneration to these soft tissue structures, enabling the meniscus to be more robust, which can result in less pain during walking (Bagwell et al., 2018). Results obtained in this study may help us in finding a suitable aPRP injection approach that can be applied in treating patients with moderate to severe degrees of knee OA.
1. Thickness of the supra-patellar bursa was measured to be > 2 mm (mm) as confirmed by musculoskeletal ultrasound. 2. Patients had history of receiving knee joint steroid injections, physical modality treatments, and taking oral NSAIDs but without any obvious reduction in SF volumes and knee pain. 3. Protruded medial meniscus causing MCL sprain as confirmed by musculoskeletal ultrasound. 4. Increased hypoechoic changes at the pes anserinus complex, indicating inflammation of the tendons and bursitis, as confirmed by musculoskeletal ultrasound. 5. The major cause of the supra-patellar bursitis, protruded meniscus with bulging MCL, and pes anserine inflammation was due to knee OA, not caused by infectious or inflammatory knee disorders. 6. Ultrasound confirmed that the supra-patellar bursa was in communication with the synovial cavity of the knee joint. This was to rule out the diagnosis of isolated cystic lesion at the supra-patellar region. 2.1. Treatment protocol The “REGEN” environmental chamber for storage of platelet concentrate kit (REGENLAB, RegenACR-C Classic, Switzerland) was used for the preparation of aPRP. After harvesting 10 mL of blood from the patient, it was then injected into the PRP test tube. Centrifugation was then performed for 15 min under the speed of 3400 rounds per minute (rpm). Approximately 6 mL of supernatant (PRP) was then aspirated from the test tube and was ready to be used for injection. The recruited patients were divided into 2 groups. Group 1 (n = 15) received 4 mL of aPRP injection to the knee joint only. Groups 2 (n = 15) received 4 mL of aPRP injection into the knee joint, and 2 mL of aPRP injection to the pes anserinus complex. Adjustment of needle depth and bevel may be needed in order for aPRP to effectively bath the pes anserine bursa, tendons (gracilis, sartorius, and semitendinosus), MCL, and the medial meniscus (Fig. 1). Musculoskeletal ultrasound was used to accurately guide the needle into the supra-patellar bursa site for the aspiration of SF, intra-articular injection of aPRP, and also injection of aPRP to the pes anserinus
2. Materials and methods In this study, a total of 30 elderly patients diagnosed with at least grade 2 on the Kellgren and Lawrence system for classification of osteoarthritis of knee and with supra-patellar bursitis were recruited into 2
Experimental Gerontology 119 (2019) 1–6
7.84 ± 2.84a,b
9.85 ± 2.78
complex. Injection was performed on a once per month basis for a total of three months. The Tablet Ultrasound Imaging System T3300 ultrasound system with a bandwidth of 4–15 MHz linear transducer was used (BenQ Medical Technology Corporation, Taipei). Accurate placement of the needle into the bursa can avoid poking into the muscle or other soft tissues that may cause blood contamination in the SF. The standard lateral approach with the knee extended was applied for ultrasound-guided SF aspiration and IA injection. The interpretation of ultrasound images and the injection procedures were done by the same physician to avoid experimental bias.
6.46 ± 2.66 7.11 ± 2.47
a,b
The proteomic technique of 2-DE was used to detect significant increases and decreases in protein spot intensities before and after PRP injections. Steps of 2-DE and subsequent protein identification using mass spectrometry (MS) can be read in our previous published article (Chen et al., 2014). SYPRO Ruby stained SF 2-DE gel images were obtained from each patient. Triplicate gel images were constructed for each patient's SF sample gathered before the 1st, 2nd, 3rd aPRP injections, and 1, 3, and 6 months after the completion of aPRP injections. The ProXPRESS™ 2D Proteomic Image System (PerkinElmer Life and Analytical Sciences) was used to scan the SYPRO Ruby stained SF 2-DE gels. The gel images were acquired as digital TIF files and analyzed using the PDQuest Basic 8.0.1 Analytical software (BioRad).
Values expressed as mean ± standard deviation (SD). a Statistical comparison between the time of treatment (or time of follow up after the completion of all 3 PRP injections) and the time of 1st PRP injection (p < 0.01). b Statistical comparison between the time of treatment (or time of follow up after the completion of all 3 PRP injections) and the time of 2nd PRP injection (p < 0.01).
13.92 ± 2.51 14.71 ± 3.44
6.88 ± 2.11
a,b
8.97 ± 2.59 10.02 ± 2.48 9.78 ± 2.61 12.76 ± 2.89 13.97 ± 2.96
a,b
23.75 ± 3.14a,b 24.97 ± 3.88a,b 32.62 ± 3.71 34.49 ± 4.07
25.54 ± 2.74a,b
27.14 ± 3.11a,b 25.72 ± 3.13a,b 33.57 ± 2.58 35.38 ± 3.78
Average SF Total Protein Concentration In μg/μL (Group 1) Average SF Total Protein Concentration In μg/μL (Group 2) Lequesne Functional Index (Group 1) Lequesne Functional Index (Group 2)
Measured parameters
1 month after the completion of all 3 monthly PRP injections Time of 3rd PRP injection (1 month after the 2nd PRP injection) Time of 2nd PRP injection (1 month after the 1st PRP injection) Time of 1st PRP injection
26.47 ± 2.23a,b
3 months after the completion of all 3 monthly PRP injections
2.2. 2-Dimension electrophoresis (2-DE)
Experimental time periods
Table 1 Differences in synovial fluid protein concentrations, and LeQuesne Index before and after aPRP injections.
26.28 ± 3.69a
25.86 ± 3.88a,b
6 months after the completion of all 3 monthly PRP injections
C.P.C. Chen et al.
2.3. Western immunoblotting Western immunoblotting method was used to validate the protein spots that revealed significant increases or decreases in concentrations after 2-DE analyses. Polyclonal and monoclonal antibodies were purchased from the ABCAM company (ABCAM, Cambridge, MA, USA) to detect the proteins of apolipoprotein A-I (APOA1), complement 5 (C5), haptoglobin (HPT), immunoglobulin kappa chain (IGKC), matrilin, MATNtransferrin (TRFE), transthyretin (TTR), and matrix metalloproteinase (MMP). Experimental steps of Western immunoblotting can be seen in our previous published article (Chen et al., 2014). The protein band intensities were analyzed using the ChemiCapture Software. 2.4. Calculation of synovial fluid total protein concentrations The standard curve of known bovine serum albumin (BSA) (Sigma, > 96% purity) concentrations (2 μg, 1 μg, 0.5 μg, 0.25 μg, and 0.125 μg) was constructed to calculate the total protein concentration of SF samples. The Bradford method was applied for the calculation of protein concentrations (Olson, 2016). 2.5. Lequesne Index The Lequesne Functional Index for degenerative knee joint was used to evaluate the extent of knee pain, and the changes of knee functional status before and after PRP injections. The Lequesne Functional Index was evaluated by the same experimenter to prevent inter-tester variability. Index value > 7 is highly indicative of degenerative knee disorder (da Silva et al., 2015). 2.6. Statistical analysis The Wilcoxon signed-rank test was used to compare the age difference between patients. The statistical tool of repeated measures ANOVA with Bonferroni correction was used to compare the SF total protein and individual protein concentrations, and changes in Lequesne Functional Index values before and after aPRP injections. Protein concentrations, and functional index values were calculated and measured at the time periods of the 1st, 2nd, 3rd aPRP injections. The follow up time periods were 1, 3 and 6 months after the completion of all 3 aPRP 3
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
validations were performed before the time of the first PRP injection and one month after the completion all 3 monthly PRP injections.
injections. The Statistical Program for Social Sciences (SPSS) program (SPSS Inc., Chicago) was used for data calculations. Values with p < 0.01 were considered statistically significant.
4. Discussion 3. Results In most of the studies related to knee OA injection treatment using aPRP, injection is done mainly into the knee joints only. Simultaneous aPRP injection into the knee joint and to the perimeniscal soft tissue structures such as the MCL, medial meniscus, and pes anserinus are seldom reported. Studies related to aPRP injection to the perimeniscal soft tissue structure such as MCL are mostly related to sport injuries or traumatic events (Bagwell et al., 2018). The efficacy of aPRP in treating patients with moderate to severe degrees of knee OA remains controversial. Although aPRP has been shown to be effective in treating patients with early and mild knee OA, it is recommended that at least 2 monthly injections are needed in order for the effect of aPRP to be more robust (Chen et al., 2017). Musculoskeletal ultrasound can be used as a tool in accurately guiding the needle to the target site that is to be treated (Chen et al., 2017). The injection needle can be accurately guided to the pes anserine bursa, tendons, MCL and the medial meniscus, and administer aPRP solution to these sites. It is technically feasible to inject to these areas under one injection spot. In other words, multiple insertion and retraction needle motions on several injection spots can be avoided using the ultrasound guidance. For instance, needle can be guided to the pes anserine tendons, then by adjusting the bevel, the needle can also be placed at the MCL for further aPRP injections, all under one needle insertion site. SF total protein concentrations in both groups revealed significant decreases after the second monthly PRP injections. Group 2 was associated with at least two-fold concentration decreases in the proteins of APOA1, MMP, IGKC, HPT, and TRFE. In SF, abundant proteins of albumin and immunoglobulin constitute about 60 to 80% of the total protein concentration (Chen et al., 2014). Therefore, significant decrease in the protein concentration of immunoglobulin may be the main cause of decreased SF total protein concentration after aPRP injections. Although the extent of inflammation in the synovial membrane of knee OA is not as obvious as in rheumatoid arthritis (RA), it is a likely ongoing process in knee OA as cartilage destruction can be observed (Chang et al., 2009). APOA1 is a plasma derived high density apolipoprotein that plays a key role in the transport of lipids. Dysregulated lipid profile in the SF is an underlying cause of inflammation in the knee joint cavity and
The mean age of the 30 recruited patients (> 65 years of age, 18 female and 12 male patients) was 73 ± 3.4 years. Wilcoxon signedrank test revealed no significant statistical differences in their age. In group 1, the average SF total protein concentrations were calculated to be 35.38 ± 3.78 μg/μL (group 2 = 34.49 ± 4.07 μg/μL) at the time of 1st aPRP injection, 33.57 ± 2.58 μg/μL (group 2 = 32.62 ± 3.71 μg/ μL) at the time of 2nd aPRP injection, 25.72 ± 3.13 μg/μL (group 2 = 24.97 ± 3.88 μg/μL) at the time of 3rd aPRP injection, and 27.14 ± 3.11 μg/μL (group 2 = 23.75 ± 3.14 μg/μL) at 1 month, 26.47 ± 2.23 μg/μL (group 2 = 25.54 ± 2.74 μg/μL) at 3 months and 25.86 ± 3.88 μg/μL (group 2 = 26.28 ± 3.69 μg/μL) at 6 months after the completion of all 3 aPRP injections. Significant decreases in SF total protein concentrations were observed after receiving 2 PRP injections in both groups (p < 0.01) (Table 1). There were no significant changes in SF total protein concentrations in both groups after receiving only the 1st PRP injection. In terms of average Lequesne Functional Index, group 1 was calculated to be 13.97 ± 2.96 (group 2 = 14.71 ± 3.44) at the time of 1st aPRP injection, 12.76 ± 2.89 (group 2 = 13.92 ± 2.51) at the time of 2nd aPRP injection, 9.78 ± 2.61 (group 2 = 7.11 ± 2.47) at the time of 3rd aPRP injection, and 10.02 ± 2.48 (group 2 = 6.46 ± 2.66) at 1 month, 8.97 ± 2.59 (group 2 = 6.88 ± 2.11) at 3 months, and 9.85 ± 2.78 (group 2 = 7.84 ± 2.84) at 6 months after the 3rd PRP injection. Significant decreases in Lequesne Functional Index were observed at the time of the 3rd PRP injection (after the completion of 2 monthly PRP injections), and in all the follow up time periods (p < 0.01) (Table 1). The 2-DE gel images of SF samples revealed at least 2-fold increases in protein intensities of matrilin, transthyretin, and complement 5 in both groups. Group 2 revealed significant decreases in the protein concentrations of haptoglobin, immunoglobulin kappa chain, transferrin, apolipoprotein A-I, and matrix metalloproteinase for at least 2fold. Decreases in the concentrations of these proteins were not significant in group 1. These protein intensities were further validated by Western immunoblotting (Table 2 states the names of SF proteins confirmed by MS and Fig. 2 reveals the images of proteins that were identified by 2-DE). Western immunoblotting protein intensity
Table 2 Protein names confirmed by mass spectrometry after 2 – DE separation and concentration differences validated by Western immunoblotting. Spot namea
Description
Access Noa
Mr (kDa)b
pIb
No. of matchedc
Seq cov (%)d
TTR MATN C5 APOA1 MMP IGKC HPT TRFE
Transthyretin ↑+ Matrilin ↑+ Complement 5 ↑+ Apolipoprotein A-1 ↓⁎ Matrix metalloproteinase ↓⁎ Immunoglobulin kappa chain ↓⁎ Haptoglobin ↓⁎ Transferrin ↓⁎
P02766 P21941 P06684 P02647 P14780 P01834 P00738 P02787
15.87 54.00 112.75 30.00 92.00 11.61 45.80 79.29
5.52 8.31 8.90 5.56 5.70 9.10 6.13 6.81
10 36 18 13 17 47 20 24
73 51 67 46 38 66 44 36
↑: increased in concentration. ↓: decreased in concentration. a Protein name and accession number according to the SwissProt and TrEMBL databases. b Predicted molecular weights in kiloDaltons and pI according to protein sequence and Swiss-2DPAGE databases. c Number of peptide masses matching the top hit from MS-Fit PMF. d Aminoacidic sequence coverage for the identified proteins. + Western immunoblotting protein spot intensity validation comparison conducted between the time of first PRP injection and one month after the completion of all 3 monthly PRP injections in both groups (p < 0.01). ⁎ Western immunoblotting protein spot intensity validation comparison conducted between the time of first PRP injection and one month after the completion of all 3 monthly PRP injections in group 2 (p < 0.01). 4
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
Fig. 2. SYPRO Ruby stained 2-DE gel image of the SF sample. The protein spot names and their molecular weights (MW) in kiloDaltons (kDa) were confirmed by mass spectrometry.
increased chondrocyte hypertrophy is observed, which is associated with cartilage degeneration. As a result, increased matrilin concentration after PRP injections may inhibit chondrocyte hypertrophy, and further attenuates cartilage degeneration (Yang et al., 2014). The complement system complements the ability of antibodies and phagocytic cells to clear pathogens. C5 is associated with degenerative joint disorders as its activation is abnormally high in human OA joints (Wang et al., 2011). Our study revealed similar findings. Therefore, increased C5 concentration may suggest improved clearance of pathogens associated with knee OA. APOA1, MMP, IGKC, HPT, and TRFE revealed significant decreases in concentrations in group 2 only. A logical way to explain this phenomenon would be that in patients with moderate to severe degrees of knee OA, joint space narrowing and perimeniscal soft tissue inflammation exist. Although the rheology of SF has changed after aPRP injections (ex/protein concentration associated with inflammation decreased and protein concentration associated with chelation increased), friction of the narrowed knee joint, and inflammation of the perimeniscal soft tissue such as the pes anserine tendons and MCL are constantly occurring. This may be the reason why group 1 patients still experience knee pain after numerous IA aPRP injections, and the decrease in the concentrations of these proteins did not reach statistical significance. After the perimeniscal soft tissue structures are injected with aPRP, the injured MCL can be healed, and the degree of inflammation at pes anserine tendons and bursa can be decreased, ensuring less laxity of these structures (Mastrangelo et al., 2011). This explains why knee pain and functional status are significantly improved when aPRP are injected both into the knee joint and to the perimeniscal soft tissue structures. In summary, results in this study revealed that the injection of aPRP can be beneficial for patients with moderate to severe degrees of knee OA. But it is strongly recommended that aPRP should be simultaneously injected into the knee joint, and also to the perimeniscal pes anserinus. Then with the adjustment of needle depth and bevel, aPRP can bath the soft tissue structures of pes anserine tendons, bursa, MCL, and medial meniscus. Multiple monthly injections are recommended in order for
induces the development of OA. The pro-inflammatory property of APOA1 was confirmed in an in vitro environment and closely associated with the protein matrix metalloproteinases (MMPs). APOA1 plays an important role in inducing strong MMP expressions (Oliviero et al., 2009). MMPs can aggravate inflammation in OA by degrading extracellular matrix macromolecules and decreasing expression of chondrocyte proteins, resulting in severe joint pain, loss of movement, and irreversible dysfunction (Lu et al., 2015). As a result, decreases in APOA1 and MMP concentrations after aPRP injections may attenuate knee joint inflammatory responses. IGKC is one of the two types of light chains that is involved in antibody production as secreted by B lymphocytes. Increased levels of free Ig light chains are detected in inflammation (Chang et al., 2009). HPT is a major acute phase glycoprotein with a molecular weight of 43 kDa. When the concentration of HPT increases by twofold or more in the SF, it is correlated with conditions such as inflammation, injury or malignancy (Park et al., 2013). TRFE is an iron-binding plasma glycoprotein that is responsible with the control of free iron in biological fluids. A study has shown that transferrin, immunoglobulin, and albumin protein concentrations are increased in OA and RA joints (Izai et al., 1992). Therefore, significant decreases in IGKC, HPT, and TRFE proteins suggest that the extent of knee joint inflammation in OA patients is decreased after aPRP injections. Three SF proteins revealed significant increases in concentrations for > 2-folds in both groups after the completion of aPRP injections. The deposition of amyloid is a risk factor in the development of OA, and increased amyloid concentration can be observed in OA knee joints. TTR protein can chelate amyloid and further preventing the deposition of amyloid in knee joints. TTR is a homotetrameric protein synthesized mainly in the choroid plexus and liver, and can be detected predominantly at the cartilage surface of aged normal cartilage samples (Akasaki et al., 2015). Therefore, significant increases in SF TTR concentrations after aPRP injections may signify a healthier knee joint as more amyloid deposits can be chelated. Matrilin protein is an extracellular matrix protein that is believed to play a crucial regulatory role in maintaining cartilage microenvironment. In matrilin knock-out mice, 5
Experimental Gerontology 119 (2019) 1–6
C.P.C. Chen et al.
the treatment of aPRP to be effective. Results in this study showed that at least two monthly aPRP injections are needed in order to achieve significant proteomic changes, and improvements in pain and functional scales. In future related research, our goal will be to conduct studies with larger sample sizes, hoping that more SF protein biomarkers related to knee OA can be discovered. Other simultaneous injection techniques will also be studied and applied, such as injecting aPRP to the genicular nerves, and to observe whether the effect of aPRP can be potentiated in patients with moderate to severe degrees of knee OA.
Chen, C.P., Hsu, C.C., Pei, Y.C., Chen, R.L., Zhou, S., Shen, H.C., Lin, S.C., Tsai, W.C., 2014. Changes of synovial fluid protein concentrations in supra-patellar bursitis patients after the injection of different molecular weights of hyaluronic acid. Exp. Gerontol. 52, 30–35. Chen, C.P.C., Cheng, C.H., Hsu, C.C., Lin, H.C., Tsai, Y.R., Chen, J.L., 2017. The influence of platelet rich plasma on synovial fluid volumes, protein concentrations, and severity of pain in patients with knee osteoarthritis. Exp. Gerontol. 93, 68–72. Cohen, M.M., Altman, R.D., Hollstrom, R., Hollstrom, C., Sun, C., Gipson, B., 2008. Safety and efficacy of intra-articular sodium hyaluronate (Hyalgan) in a randomized, double-blind study for osteoarthritis of the ankle. Foot Ankle Int. 29, 657–663. da Silva, F.S., de Melo, F.E., do Amaral, M.M., Caldas, V.V., Pinheiro, I.L., Abreu, B.J., Vieira, W.H., 2015. Efficacy of simple integrated group rehabilitation program for patients with knee osteoarthritis: single-blind randomized controlled trial. J. Rehabil. Res. Dev. 52, 309–322. de Miguel Mendieta, E., Cobo Ibanez, T., Uson Jaeger, J., Bonilla Hernan, G., Martin Mola, E., 2006. Clinical and ultrasonographic findings related to knee pain in osteoarthritis. Osteoarthr. Cartil. 14, 540–544. Gorg, A., Weiss, W., Dunn, M.J., 2004. Current two-dimensional electrophoresis technology for proteomics. Proteomics 4, 3665–3685. Izai, M., Miyazaki, S., Murai, R., Morioka, Y., Hayashi, H., Nishiura, M., Miura, K., 1992. Prorenin-renin axis in synovial fluid in patients with rheumatoid arthritis and osteoarthritis. Endocrinol. Jpn. 39, 259–267. Khoshbin, A., Leroux, T., Wasserstein, D., Marks, P., Theodoropoulos, J., Ogilvie-Harris, D., Gandhi, R., Takhar, K., Lum, G., Chahal, J., 2013. The efficacy of platelet-rich plasma in the treatment of symptomatic knee osteoarthritis: a systematic review with quantitative synthesis. Arthroscopy 29, 2037–2048. Kon, E., Mandelbaum, B., Buda, R., Filardo, G., Delcogliano, M., Timoncini, A., Fornasari, P.M., Giannini, S., Marcacci, M., 2011. Platelet-rich plasma intra-articular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy 27, 1490–1501. Laudy, A.B., Bakker, E.W., Rekers, M., Moen, M.H., 2015. Efficacy of platelet-rich plasma injections in osteoarthritis of the knee: a systematic review and meta-analysis. Br. J. Sports Med. 49, 657–672. Lu, S., Xiao, X., Cheng, M., 2015. Matrine inhibits IL-1beta-induced expression of matrix metalloproteinases by suppressing the activation of MAPK and NF-kappaB in human chondrocytes in vitro. Int. J. Clin. Exp. Pathol. 8, 4764–4772. Lueders, D.R., Smith, J., Sellon, J.L., 2016. Ultrasound-guided knee procedures. Phys. Med. Rehabil. Clin. N. Am. 27, 631–648. Mastrangelo, A.N., Vavken, P., Fleming, B.C., Harrison, S.L., Murray, M.M., 2011. Reduced platelet concentration does not harm PRP effectiveness for ACL repair in a porcine in vivo model. Journal of Orthopaedic Research: Official Publication of the Orthopaedic Research Society. 29, 1002–1007. Oliviero, F., Sfriso, P., Baldo, G., Dayer, J.M., Giunco, S., Scanu, A., Bernardi, D., Ramonda, R., Plebani, M., Punzi, L., 2009. Apolipoprotein A-I and cholesterol in synovial fluid of patients with rheumatoid arthritis, psoriatic arthritis and osteoarthritis. Clin. Exp. Rheumatol. 27, 79–83. Olson, B.J., 2016. Assays for determination of protein concentration. Curr. Protoc. Pharmacol. 73 A.3a.1-a.3a.32. Park, H.J., Oh, M.K., Kim, N.H., Cho, M.L., Kim, I.S., 2013. Identification of a specific haptoglobin C-terminal fragment in arthritic synovial fluid and its effect on interleukin-6 expression. Immunology 140, 133–141. Uysal, F., Akbal, A., Gokmen, F., Adam, G., Resorlu, M., 2015. Prevalence of pes anserine bursitis in symptomatic osteoarthritis patients: an ultrasonographic prospective study. Clin. Rheumatol. 34, 529–533. Wang, P., Yang, L., Li, H., Lei, Z., Yang, X., Liu, C., Jiang, H., Zhang, L., Zhou, Z., Reinhardt, J.D., He, C., 2016. Effects of whole-body vibration training with quadriceps strengthening exercise on functioning and gait parameters in patients with medial compartment knee osteoarthritis: a randomised controlled preliminary study. Physiotherapy 102, 86–92. Wang, Q., Rozelle, A.L., Lepus, C.M., Scanzello, C.R., Song, J.J., Larsen, D.M., Crish, J.F., Bebek, G., Ritter, S.Y., Lindstrom, T.M., Hwang, I., Wong, H.H., Punzi, L., Encarnacion, A., Shamloo, M., Goodman, S.B., Wyss-Coray, T., Goldring, S.R., Banda, N.K., Thurman, J.M., Gobezie, R., Crow, M.K., Holers, V.M., Lee, D.M., Robinson, W.H., 2011. Identification of a central role for complement in osteoarthritis. Nat. Med. 17, 1674–1679. Yang, X., Trehan, S.K., Guan, Y., Sun, C., Moore, D.C., Jayasuriya, C.T., Chen, Q., 2014. Matrilin-3 inhibits chondrocyte hypertrophy as a bone morphogenetic protein-2 antagonist. J. Biol. Chem. 289, 34768–34779. Yoon, H.S., Kim, S.E., Suh, Y.R., Seo, Y.I., Kim, H.A., 2005. Correlation between ultrasonographic findings and the response to corticosteroid injection in pes anserinus tendinobursitis syndrome in knee osteoarthritis patients. J. Korean Med. Sci. 20, 109–112.
5. Conclusion This study aimed to examine whether the injection of aPRP is effective for patients with moderate to severe degrees of knee OA. Results in this study showed that in the group of patients who received simultaneous aPRP injection intra-articularly and to the perimeniscal soft tissue of pes anserine, pain and functional scores were significantly improved. The concentrations of SF total protein, and proteins associated with inflammation (eg/APOA1, MMP, IGKC, HPT, and TRFE) were significantly decreased. Protein concentrations associated with chelation and anti-aging physiological functions (eg/TTR, MATN, and C5) were increased significantly. As a result, the rheology of knee SF appears to become less susceptible to degeneration after aPRP injections. As a result, at least 2 monthly injection of IA aPRP in conjunction with accurate injection of aPRP to the perimeniscal soft tissue structure such as the pes anserinus may be a viable option in treating patients with moderate to severe degrees of knee OA. Conflict of interest All the authors in this study have no conflict of interest with any financial organization regarding the material discussed in the manuscript. Acknowledgements This study was supported by the grants from the National Science Council, Taiwan (NMRPG3F6261-2 (NMRPD1F1642), 105-2314-B182A-023-MY2) and the Chang Gung Memorial Hospital at Linkou Research Project Grants of CMRPG1F0091-2 to Dr. Carl P.C. Chen. The National Science Council grant supported the expenses of consumable products. The Chang Gung Memorial Hospital Research Project Grant supported the cost of western immunoblotting analyses and the purchase of antibodies. References Akasaki, Y., Reixach, N., Matsuzaki, T., Alvarez-Garcia, O., Olmer, M., Iwamoto, Y., Buxbaum, J.N., Lotz, M.K., 2015. Transthyretin deposition in articular cartilage: a novel mechanism in the pathogenesis of osteoarthritis. Arthritis Rheum. 67, 2097–2107. Bagwell, M.S., Wilk, K.E., Colberg, R.E., Dugas, J.R., 2018. The use of serial platelet RICH plasma injections with early rehabilitation to expedite grade iii medial collateral ligament injury in a professional athlete: a CASE report. Int. J. Sports Phys. Ther. 13, 520–525. Chang, X., Cui, Y., Zong, M., Zhao, Y., Yan, X., Chen, Y., Han, J., 2009. Identification of proteins with increased expression in rheumatoid arthritis synovial tissues. J. Rheumatol. 36, 872–880.
6