Activation is carried out with only autologous thrombin, in the proportion of 0. Descriptive analyses are provided for demographic and clinical characteristics.
The Kolmogorov-Smirnov and Shapiro-Wilk tests were applied to check distributions for normality. Chi-square tests were used to compare binary outcomes and the, Kruskall-Wallis and Wilcoxon tests were applied to compare distributions of continuous data The primary analysis is the median change in VAS and WOMAC scores from pre-treatment baseline to days across the three groups. A two-sided p value less than 0. The mean age was In all of the patients, a mild adverse reaction in the form of a knee swelling was reported days after the application.
It was not a reported as a major complication by any patient. It was verified that the majority of patients in all the groups have grade II of OA by Kellgren-Lawrence in the right knee.
Also, it was observed that the patients were overweight through BMI in all the groups of treatment. The majority of the population was brown and has as comorbidities hypothyroidism, dyslipidemia and hypertension. Half of the patients in all the groups were physically active, practicing walking or aquatic activities, without axial impact.
These data were presented in Table 1. This tendency was verified at days, as demonstrated in Table 6. Relative to PRP, our final PRP product consisted of platelets, leukocytes and circulating fibrinogen, with a small residue of red cells Figure 2.
The PRP platelet concentration varied between , and 1,, per mm 3 of plasma, which corresponds to 5 to 8 times the basal concentration in all patients. The basal concentration of platelets was In relation to the inflammation, the level of CPR showed evidence that the group treated with PRP alone or in association had an increase in the CPR levels after 90 days and a decrease at the end of the follow-up.
On the other hand, the group treated with HA showed an increase in 30 days with a posterior decrease in 90 days, but at the end of follow-up the levels of CPR were higher than baseline, as verified in the Table 7.
In and days after treatment it was verified the same pattern. The use of buffy-coat or leukocyte layer together with PRP was incentivized by studies that highlighted the antimicrobial [ 25 , 26 ] and immune-regulatory [ 27 , 28 ] actions of the leukocytes, as well as proving that the majority of the platelets are found in this layer, together with the leukocytes after the centrifugation [ 29 ]. There are controversies as to the use of the leukocytes as studies suggests that with the presence of leukocytes, the neutrophils are enabled to liberate metalloproteins that cause degradation to the extracellular matrix and even release free radicals [ 30 ].
However, the macrophages are responsible for the removal of the debris, phagocytic function and also have an important role in the balance of the pro-inflammatory and anti-inflammatory aspects of healing. As it is not possible to fractionate the different types of white blood cells, it may be that the absence of macrophages could be more harmful to the cure than any secondary harm inflicted by the presence of the neutrophils.
We believe that this increase in CPR levels in the first 90 days of the treatment in groups treated with PRP can be explained due to the increase of inflammatory activity in the joint because of the WBC.
Relative to activation of PRP, the use of chemical activators cause instability in the fibrin network and rapid growth factor release. On the other hand, if the PRP is activated in a more physiological manner, a stable tetramolecular network is formed and it has direct influence in the speed and amount of liberation of the growth factors [ 31 ]. Recently, there has been a lot of interest in new treatments aimed at stimulating repair or replacing damaged cartilage in joints.
There are currently limited high-level studies in the literature to demonstrate the real efficacy of PRP injections. Sampson et al [ 32 ] presented a pilot study involving 14 patients with primary and secondary knee OA where treatment with PRP resulted to significant improvement in function and relief of pain and symptoms.
Most of the patients expressed favorable outcome at 12 months after treatment. Kon et al [ 33 ] published a study involving patients with chronic degenerative condition of the knees treated with intraarticular knee injections and followed at 6 and 12 months. They showed statistically significant improvement in all clinical scores and concluded that their preliminary results indicate that treatment with PRP injections is safe and has the potential to reduce pain and improve knee function and quality of life in younger patients with low degree of articular degeneration.
Sanchez et al [ 34 ] performed an observational cohort study of 30 patients with knee OA and showed significant reduction of the WOMAC pain subscale at 5 weeks for the autologous preparation rich in growth factors PRGF group.
In our study, the patients enrolled have some significant differences between the groups when evaluated the race and comorbidities. In relation to race, it was verified that in the group that used association of PRP and HA, we had more black and less Caucasian people than the other groups. However, in Brazil we have extreme population miscegenation and arthritis does not have relevance in context with race. Kon et al [ 35 ] showed that the PRP group showed better results than the HA group at 6 months follow up in the International Knee Documentation Committee and VAS scores and concluded that autologous PRP injections showed more and longer efficacy than HA injections in reducing pain and symptoms and recovering articular function.
It was verified that the groups treated with a single or double injection of PRP had an improvement in relation of placebo, however, in 6 months occurred the deterioration of the results. Our study showed an improvement even after 1 year and this can be explained due to one more application and the use of PRP rich in leukocytes.
Many studies have suggested that the application of hyaluronic acid and PRP may have potentially positive effects on cartilage repair and slow down the progression of OA [ 9 — 11 ].
However, to our knowledge, there is lack of studies in literature that examined the combination use of HA and PRP. Studies show that HA provides appropriate matrix and supportive scaffold material for cartilage repair and enhances the mechanical properties of the cartilage [ 37 — 39 ]. Thus, it is hypothesized that their combination may be synergistic. According to literature, combining PRP and HA may benefit from their dissimilar biological mechanisms and helping with the signaling molecules as inflammatory molecules, catabolic enzymes, cytokines and growth factors.
The time to all these modifications happen is between days after application, which explains the days of improvement in WOMAC PA [ 40 , 41 ].
The lubrication and support to the extracellular matrix that the HA provided seemed to enable earlier functional benefit to the PRP injection. This combination may result to better rehabilitation and earlier return to activities of daily activities. When the inflammation was evaluated, it was verified that the groups that treated with PRP alone or in combination presented lower levels of CPR at the end of follow-up in comparison to HA.
Interestingly, in these groups an increase in CPR levels was observed at 90 days. This increase could be explained due to the inflammatory process of cells in the regenerative phase of treatment and also peak of improvement in treatments based in cell therapy. The study utilized self-reported questionnaires such as WOMAC and VAS to assess pain and functional outcomes, which could potentially limit the objectivity of results.
In addition, advanced imaging such as MRI was not performed because of high costs, but could have provided more objective data as to the benefit of treatments. Although functional tests can provide more objective responses to treatment, they were not included in the study and should be considered for future studies.
Another limitation was the absence of a gold standard or true control group using saline. Our results suggest that the use of autologous PRP and its combination with HA are safe and effective methods for treatment of mild to moderate osteoarthritis of the knee.
Combining HA and PRP resulted to significantly less pain and less functional limitation compared to HA alone up to 1 year after treatment.
More randomized controlled studies with larger numbers of patients are needed to confirm these findings and to investigate the persistence of the beneficial effects observed. The authors have no conflicts of interest and received no sponsorship for any of the products used in the trial. National Center for Biotechnology Information , U.
J Stem Cells Regen Med. Published online Nov Lana , 1, 4 Adam Weglein , 3 Steve E. Sampson , 2 Eduardo F. Souza , 4 Mary A. Onodera , 7 Joyce M. Annichino-Bizzacchi , 7 Maria Helena A. Santana , 8 and William D. Belangero 4. Lana 1. Adam Weglein 3. Steve E. Sampson 2. Eduardo F. Vicente 1. Stephany Cares Huber 1. This is a parameter measured or observed that is recorded at a defined time and can be assumed to reflect the effect of a treatment. The endpoint may be clinical, e. In a confirmatory study hypotheses are formulated a priori according to the primary study question.
If the primary objective of the trial is to demonstrate the superiority of a new treatment over an existing treatment or placebo, then the initial assumption null hypothesis is that the two treatments do not differ in efficacy. Based on statistical analysis the null hypothesis can be retained or must be rejected in favor of the alternative hypothesis.
The alternative hypothesis is assumed when a statistically significant difference is ascertained between the two treatments. A detailed description of methods for statistical evaluation is given in an earlier article in this series [ 15 ].
The primary study question is accompanied by one or more ancillary study questions, i. The secondary endpoints investigate other effects of the treatment, e. In the ALIFE study, the secondary endpoints included the rate of miscarriage, the premature birth rate, and the rate of maternal thrombopenia. From the statistical viewpoint it is vital to distinguish between the primary and secondary study questions, because the number of study subjects depends solely on the primary endpoint Study planning includes calculation of the number of subjects necessary for detection by statistical analysis of a minimally relevant difference in efficacy, from the clinical viewpoint, between the treatments.
The number of patients is therefore crucial for the statistical power of a study. Sample size calculation is described in detail in a previous article in this series [ 17 ]. In order to demonstrate the postulated positive effect of the combination therapy, women were enrolled in the trial. In trials with randomized and controlled design e. The patients in the control group receive either another treatment or a placebo. The ALIFE trial is a three-armed parallel group study to establish whether the combination treatment or the monotherapy improve the live birth rate compared with placebo.
The use of placebos in clinical trials is ethically justified provided that no standard treatment is available. If comparison with placebo is indispensable for methodological reasons, it can be justified as long as patients will not be harmed That is the case, for example, if the study is of only short duration or if the severity of disease permits postponement or interruption of treatment. As in any study of human subjects, the study population of an RCT must be clearly defined.
Precise inclusion and exclusion criteria are elaborated to ensure that only eligible patients are recruited. The study participants must be homogeneous with regard to demographic characteristics, disease state, and possibly even comorbidity and comedication. This can be achieved by standardization of, for example, the time s of intake of the study medication and the methods used to measure clinical parameters, but most important for comparability is randomization of the participants.
In RCTs the patients are randomly assigned to the different study groups. This is intended to ensure that all potential confounding factors are divided equally among the groups that will later be compared structural equivalence. Only if the groups are structurally equivalent can any differences in the results be attributed to a treatment effect rather than the influence of confounders. If the confounders are known, structural equivalence of the patient groups can be attained by stratified randomization Box.
In the ALIFE study the patients were assigned to the three treatment groups with a randomization ratio of If patients were allocated to treatment groups by conscious or unconscious selection for prognosis-related characteristics, rather than randomly, this could lead to biased treatment comparison and distorted results selection bias. The assignment to study groups must not be in any way predictable.
Predictability of group allocation is avoided by ensuring the study staff are unaware to which treatment the next patient will be allotted. Alternating assignment to the different treatments is not truly random.
Bias is avoided not only by randomization but also by blinding. A study may be double blind, single blind, or open. In a double-blind study neither patient nor study physician knows to which treatment the patient has been assigned.
Double-blind studies are advantageous if knowledge of the treatment might influence the course and therefore the results of the study. Thus it is particularly important that the study physician is blinded to treatment if the endpoints are subjective. Blinding of patients to their treatment is important, for example, if their attitude could potentially affect their reliability in taking the test medication compliance or even their response to treatment.
If only one party, either patient or study physician, is blinded to the treatment, the study is called single blind; a study with no blinding is described as open. The highest possible degree of blinding should be chosen to minimize bias.
The data subjected to statistical analysis in RCTs are those gathered from patient populations defined in the study protocol. The primary population for analysis is the so-called intention-to-treat ITT population, comprising all randomized patients. In analysis according to the ITT principle, patients are allocated to the group to which they were randomized, thus retaining the advantages of randomization such as structural equivalence. Because the ITT population includes all patients who were randomized, the data for analysis include some patients whose treatment was interrupted, prematurely discontinued, or did not take place at all.
The analysis strategy for ITT data is therefore conservative, i. Many studies define a modified ITT mITT population, which may for example comprise the patients who received at least a defined amount of study treatment.
An alternative strategy is to restrict analysis to the data from the per-protocol PP population. Patients in whom study conduct deviated from the protocol are excluded from analysis. These so-called protocol violations include, for example, failure concerning the application of inclusion or exclusion criteria and incorrect administration of the study treatment.
In analysis according to the PP principle, patients are allocated to the treatment groups depending on the treatment they actually received. Because the PP population includes only those patients who completed the study according to the protocol, the results may be distorted in favor of the investigational intervention To assess the robustness of the study findings, PP evaluation is carried out as a sensitivity analysis if the ITT population is the patient population for the primary efficacy analysis If the results of PP and ITT evaluation of the primary endpoint are very similar, they can be regarded as reliable.
Should this not be the case, the possible reasons for the discrepancy between the results of the ITT and PP analyses must be discussed in the results section of the publication. The rates of live births in the three treatment groups did not differ significantly Table 1. Analysis according to the PP principle confirmed this finding. Neither aspirin and heparin combined nor aspirin alone were demonstrated to have a greater effect than placebo on the live birth rate.
Relative risk and absolute difference were calculated for the comparisons between aspirin plus heparin and placebo and between aspirin alone and placebo.
The p-value applies to all treatment group comparisons. Conclusion We found no significant effect of treatment group for trunk muscle thickness. Participants reported a slightly greater sense of perceived recovery with the McKenzie method than with the motor control approach. Level of Evidence Therapy, level 1b-. Registered September 7, at www. J Orthop Sports Phys Ther ;46 7 Epub 12 May
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