The broad incidence of osteoarthritis of the knee has a substantial influence on people’s daily lives, resulting in functional impairment and a deterioration in the quality of life. Due to its intricate natural history, it has proved difficult to characterize, diagnose, and cure. This condition is distinguished by articular cartilage deterioration, bony border thickening, subchondral bone rigidity, and inflammatory alterations in the synovial membrane and joint capsule. It is particularly disconcerting in India, where its symptomatic prevalence is 28.7% ^[1]. Arthroplasty remains the final treatment modality of choice for end-stage osteoarthritis of the knee, whereas early stages of osteoarthritis have traditionally been treated with symptomatic relief modalities such as analgesics and many other disease-modifying and chondroprotective agents. However, these modalities provide no advantage in halting the natural course of the disease and decreasing the amount of destruction caused by the disease ^[2]. The failure of the present treatment options to provide disease-modifying effects in mild and moderate grades of knee osteoarthritis has propelled the research to find options able to provide the same. Intra-articular botulinum toxin injections, autologous chondrocyte implantation, platelet-rich plasma (PRP), plasma rich in growth factors (PRGF), and mesenchymal stem cell therapy are recent advances in the effort to discover disease-modifying treatments. The name “Advanced Therapies” (AT) has been applied to various techniques that aim to rejuvenate or restore deteriorated tissue. Unfortunately, most of these newer modalities are still out of the reach of our country’s general population for various reasons ranging from lack of infrastructure to financial constraints ^[3]. PRP has carved out a comfortable niche in the fields of regenerative musculoskeletal medicine due to its properties in repairing injured tissue and its role in degenerative diseases. Furthermore, the ease of access has made it a widely available treatment modality for knee osteoarthritis to the general population [4, 5]. Recently though, PRGF, or PRGF, has caught the eye of clinicians one and all which is a variant of PRP that is reported to have 3 times as many platelets as PRP and no white or red blood cells. It is the by-product of PRP that does not contain leucocytes or inflammatory cytokines, which is believed to be more effective and has fewer side effects than PRP [6, 7]. The major difference between PRP and PRGF preparations is that PRP contains whole platelets which require the host fibrin to cause a breakdown of these platelets at the injection site and deliver the growth factors. In contrast, PRGF as a preparation contains platelets that are already broken down due to the addition of platelet-activating factors such as calcium chloride thus delivering only growth factors at the administration site [8, 9]. Various research literature available talk about the chondroprotective and chondro-regenerative properties of both PRP as well as PRGF; however, this belief remains unsatisfactorily tested with no consensus over the presence or the extent to which these modalities bear this supposedly sought-after characteristic. Considering the problem at hand, we hypothesized that biochemically testing the biomarkers of cartilage degeneration before and after interventions with these modalities could educate us about their effect on the articular cartilage. In the field of cartilage degeneration biomarkers, serum Coll2-1 stands out as a unique and novel biomarker. Several clinical studies confirm its usefulness as an indication of disease severity, therapeutic response, and diagnosis [10-13]. Due to its extensive history of research, PRP is now well accepted as a symptomatic treatment modality for knee osteoarthritis, whereas PRGF is a relatively new version of the established PRP protocol; thus, comparative studies between the two are limited along with scarce literature explaining and quantifying the cartilage regeneration of these modalities. To that end, we compared the clinical effectiveness of PRP and PRGF and evaluated their cartilage regeneration potential by studying the change in blood biomarkers related to cartilage breakdown, Coll2-1 before and after the intervention.

Study design

Enrolled in this triple-blind comparative randomized trial were patients with mild-to-moderate osteoarthritis of the knee who visited the outpatient department of a tertiary care center in northern India between December 2022 and March 2023. The trial involved three intra-articular interventions with PRP or PRGF, each spaced 4 weeks apart, and followed up with the clinical and serological assessment of the effects rendered by the said interventions for 9 months following the third intervention. The trial began after receiving approval from the Institution’s Ethical Committee, with the registration number CTRI/2022/12/048590 by the Clinical Trials Registry India (Registration Date-December 29, 2022).

Inclusion and exclusion criteria

Patients who presented to the outpatient department of our tertiary care center with complaints of non-traumatic knee pain with a prior history of at least 3 months were examined and a diagnosis of primary knee osteoarthritis was made based on the American College of Rheumatology criteria for diagnosing knee OA ^[14]. A radiological grading of the diagnosed knee OA was done according to the Kellgren-Lawrence grade system on standing anteroposterior and lateral knee X-rays ^[15]. The KL grades 2 and 3 patients were recruited into the study after attaining written informed consent. Patients were excluded if they had previously operated knees, inflammatory arthritis, active knee infection, suffered from anemia or bleeding disorders, had angular deformities >5°, uncontrolled diabetes mellitus, ligamentous instability of the knee, history of trauma, any malignancy or, osteoarthritis of any other joint.

Recruitment and randomization

A total of 98 patients were enrolled during the study process after applying the aforementioned inclusion/exclusion criteria. Using a computer-generated random allocation technique (research randomizer form v4.0), a clinician from the department who was not participating in the study performed the randomization. Triple-blinding was kept in place by not telling the patient which modality that they were receiving (this was explained to them during the consent process), not telling the injecting clinician which injection to give, and not telling the statistician analyzing the data which group had received which injection (Fig. 1).

Sample size and statistical analysis

The National Health Portal for India’s data on the prevalence of knee osteoarthritis led to the calculation of 66 as the minimal sample size for the current investigation. For statistical analysis, the means and standard deviations of each group’s measurements were employed (SPSS 22.00 for Windows; SPSS Inc., Chicago, USA). One-way analysis of variance was used to statistically examine the data for each evaluation point. Depending on whether the data were qualitative or quantitative, the difference between the two groups was assessed using the Chi-square test or the t-test, with a significance level of P < 0.05 and a 95% confidence interval. Coll2-1 levels were compared intra-group (mean baseline vs. mean at 12-month follow-up) and inter-group (mean baseline in PRP group vs. mean baseline in PRGF group and mean at 12-month follow-up in PRP group versus mean at 12-month follow-up in PRGF group) using the Tukey HSD post hoc test

Intervention

The PRP preparation was made using 8.5 mL acid citrate dextrose (Becton, Dickinson and Company, USA) tubes through the double spin method ^[16] while PRGF was prepared using a PRGF preparation kit by the name of OSSINEXT (Wockhardt, USA). For the PRGF, 4 mL of venous blood was collected in the given vacutainer containing platelet-activating propriety solution. The blood was mixed 6–10 times gently and kept in an upright position for 30 min. The vacutainer was centrifuged at 3400 rpm for 10 min along with a counter-balance. The fleshly prepared platelet products were injected under all aseptic precautions in the affected knee of the patient. The patient was sat on a couch with their legs hanging from the side of the couch in 90° of flexion at the knee joint. The product was injected using a 22G needle through the anterolateral approach without any preceding local anesthesia. The patients were asked to actively flex and extend their knees post-injection to spread the injected product evenly within the joint. A total of 3 PRP and PRGF injections were given to the respective groups at intervals of 4 weeks each. The patients were kept under observation post-injection for about half an hour and sent to their homes post that. Only oral tramadol was advised on a needful basis, along with ice fomentation of the injection site for the next 3 days (Fig. 2).

Biomarker analysis

Cusabio’s human Coll2-1 ELISA kits (product code-CSB-EQ027311HU) were used for the evaluation of the biomarker. Before the initial injection and at the 12-month follow-up, the patient had 5 mL of blood drawn. This test utilized the sandwich ELISA test technique for quantitative analysis. The color intensity developed to the amount of Coll2-1 bound on the substrate microplates was determined to ascertain the levels of the biomarker (Fig. 3).

Follow-up and outcome analysis

Patients were called up for the initial 3 months sequentially for 3 IA injections of PRP or PRGF and then again at 6 months and 12 months from the first injection. The outcome assessment was done using patient-reported outcome scores, Visual Analog Scale (VAS) ^[17] and Western Ontario and McMaster Universities Arthritis Index (WOMAC) ^[18] at baseline before the first injection, before the second injection, before the third injection, at the 6-month follow-up and the 12-month follow-up. Furthermore, serum Coll2-1 levels were determined before the first injection to define a baseline value and then again at the 12^th-month follow-up from the first injection.

Throughout this trial, 98 patients with knee pain were recruited from the OPD after applying the defined inclusion and exclusion criteria. The recruited patients were randomized into two groups. Only 72 patients of all the recruited individuals completed the stipulated 12-month follow-up, and hence, they were the ones that were included in the final data analysis. There were 83% females in the PRGF group and 75% females in the PRP group. The mean age of the patients in the PRGF group was 58.19 ± 11.51 years and in the PRP group, it was 56.3 ± 7.24 years. The mean BMI in the PRP group was 26.21 ± 4.68 while in the PRGF group, it was 26.05 ± 3.03. The PRP group consisted of 25% of patients with grade 2 osteoarthritis and 75% of patients with grade 3 osteoarthritis, while the PRGF group had 19.4% of patients with grade 2 osteoarthritis and 80.6% with grade 3 osteoarthritis. The mean baseline serum Coll2-1 level in the PRP group was 1545.92 ± 371.86 pg/mL while it was 1398.78 ± 409.88 pg/mL in the GFC group. This distribution of the subjects based on the baseline characteristics was found to be statistically homogenous as there were no statistically significant differences between the groups (Table 1).

The difference between the baseline mean serum Coll2-1 values and the mean value obtained at the 12-month follow-up was found to be significant in both the PRP (P = 0.005) and PRGF (P = 0.009) groups showing significant reductions in the Coll2-1 levels at the 12-month follow-up of the patient. An inter-group comparison of the mean reduction (improvement) from the baseline values showed no significant reduction at the 12-month follow-up between the PRP (437.14 ± 145.27) and the PRGF group (407.67 ± 189.12). The inter-group comparison of the mean serum values of the biomarker revealed no significant difference at the 12-month follow-up between both groups (P = 0.21) (Table 2). The inter-group comparison between the two sets of data showed better VAS outcomes with a statistically significant difference in the PRGF group at the 6-month (P = 0.032) and the 12-month follow-up (P = 0.038). The improvements in VAS in both groups reached statistical significance when compared to baseline values at the 2-month, 3-month, 6-month, and 12-month follow-ups, respectively (P ≤ 0.01). The mean reduction in VAS scores (improvement) in the PRGF group was 4.14 ± 0.88 points compared to baseline values, which were significantly higher than the mean reduction in the PRP group, which was 2.96 ± 0.77 (P = 0.003) (Tables 3 and 4).

The inter-group comparison between the two sets of data showed better WOMAC outcomes with a statistically significant difference in the PRGF group after the 3^rd injection (P = 0.037), at 6 months (P = 0.032) and the 12-month follow-ups (P = 0.046). The improvements in WOMAC in both groups reached statistical significance when compared to baseline values after the 2^nd injection, after the 3^rd injection, and at the 6-month and the 12-month follow-ups, respectively (P ≤ 0.01). The mean reduction in WOMAC scores (improvement) in the PRGF group was 29.23 ± 6.53 points as compared to baseline values which were higher than the mean reduction noticed in the PRP group which was 18.86 ± 6.11 (P = 0.006); however, it was not statistically significant (Table 5 and 6).

India is a major economy developing and taking massive strides in all aspects of human advancement currently, at such an important juncture, the massive prevalence of a crippling disease like knee osteoarthritis poses a significant hurdle toward the functioning and quality of life of the people driving this uplift. Hence, it goes without saying that the need of the hour is a disease-modifying treatment modality having the reach and efficacy to reduce the prevalence of the disease in the country and also worldwide as a whole. Appreciating the problem at hand our single-center triple-blind randomized study took place in a tertiary care facility in the north of India intending to compare the clinical effectiveness of PRP and PRGF and evaluate their cartilage regeneration potential by studying the change in blood biomarkers related to cartilage breakdown, Coll2-1 before and after the intervention. Coll2-1 is a pattern of 9 amino acids (HRGYPGLDG) produced from the collagen type II alpha chain, which has proven to be a potentially helpful biomarker. Coll2-1 and Coll2-1NO2 are products of the oxidative-associated collagen type II degradation network. Serum Coll2-1 has demonstrated diagnostic value in assessing illness severity, prognosis, and therapy efficacy. Serum and urinary Coll2-1 levels have been demonstrated to predict radiographic knee joint space narrowing progression. Deberg et al. did a study to investigate the serum levels of Coll2-1 and Coll2-1 NO2 and found them to be biomarkers of oxidative-related type 2 collagen network degradation in patients with osteoarthritis, hence prompting the use of serum Coll2-1 for this study ^[19]. Our study showed significant reductions in the serum levels of Coll2-1 from the baseline values taken before the intervention in both the PRP as well as the PRGF group, suggesting that both PRP and PRGF intra-articular injections were able to reduce the disease-related wear of the knee articular that occurs in knee osteoarthritis. However, at the 12-month follow-up, there was no statistically significant difference between the mean values of both the groups, so it can be said that intra-articular injections of both PRP and GFC were equally efficacious at reducing the cartilage wear in the randomized study population, in this study, however. While there are no available studies where serum Coll2-1 levels were analyzed after IA injections of PRGF, the results of this study were in accordance with the findings of Fawzy et al. who evaluated the early change in serum levels of Coll2-1 after a single injection of PRP after 3 months and found a significant decrease in the values ^[20].  Our study revealed statistically significant reductions in VAS and WOMAC scores at the 12-month follow-up as compared to the baseline in both groups. The Minimum Clinically Important Difference (MCID) in the case of WOMAC is considered when there is a 12% decrease in values from the baseline or a 6% decrease from the maximum score, our study revealed these clinically significant reductions in WOMAC scores in both the PRGF (41% decrease) as well as the PRP (27% decrease) at the 12-month follow-up. In the case of VAS, there is not a clear consensus on the MCID in the available literature; however, it is understood to be a difference of 2 cm between 2-time points for patients with VAS more than 7. In our study, there was a mean reduction in VAS scores of 4.14 cm in the PRGF group and 2.96 cm in the PRP group at the 12-month follow-up from the baseline, suggesting a clinically significant improvement in VAS scores in both groups [21-23]. There was a significant statistical difference between both the groups concerning VAS and WOMAC scores at the 6-month and the 12-month follow-up with patients in the PRGF group showing statistically better improvements at the mentioned time points of the study, which may be due to the effect PRP vanning off in the later stages of the follow-up while the residual effect of PRGF causing greater statistical improvements in the study population. The above-mentioned observations in our study suggest that while both PRP and PRGF are efficacious at providing statistically and clinically significant improvements in the study population at the 12-month follow-up, the PRGF was better at providing these effects as early as after the 3^rd injection and also at the 6^th and 12^th-month follow-up. While there have been no studies comparing both PRP and PRGF directly in patients of knee osteoarthritis, the findings of our study are in line with those of Raeissadat et al. when they compared the effects of intra-articular injection of PRP, PRGF, hyaluronic acid (HA), and ozone in knee osteoarthritis and found that PRP and PRGF were more efficacious than the other modalities at decreasing the disease-related pain and improving function over a study period of 12 months ^[7]. While there are not many studies comparing both PRGF and PRP in knee osteoarthritis, the effect of PRP in knee osteoarthritis has been thoroughly investigated and the results of our study fall in line with various studies undertaken by Halpern et al., Cerza et al., Filardo et al., Patel et al., Chang et al., and many others, who have all reported favorable outcomes after therapy with IA PRP injections in patients of knee osteoarthritis [24-28]. Both PRP and PRGF function by delivering growth factors, mainly, platelet-derived growth factors, insulin-like growth factors, vascular endothelial growth factors, transforming growth factors and many more to the affected areas and provide a base where potential regeneration of the cartilage can occur. Both these modalities also provide anti-inflammatory effects by neutralizing the oxidative stress caused by the inflammatory cytokines and the free radicals slowing down the cartilage erosion being caused by them. The findings in our study can be laid down to the fact that PRP delivers whole platelets to the joints which breakdown in vivo to provide the growth factors, but that sometimes leads to growth factor loss as not all of the delivered platelets will break down to provide the growth factors, while, on the other hand, PRGF delivers pure growth factors derived from already broken down platelets; thus, a much higher concentration of growth factors is delivered which is available for both, providing a scaffolding for new cartilage to be laid and also acting as a reservoir of growth factors to be used up over a longer period [6, 29-31]. Although further studies building on the foundations of the results achieved in our study are required with a bigger study population and involving a more diverse group of centers, we, the authors of this study found intra-articular injections of PRGF comparatively more beneficial in reducing disease-related pain and improving joint function and capable of reducing disease-related cartilage degradation in patients of knee osteoarthritis over 12 months. A longer follow-up might well show us lasting relief and regenerative effects stretching further than the stipulated 12-month follow-up of this study while radiological co-relation using magnetic resonance imaging or ultrasonogram would also help in cementing the results of our current study.

Limitations

A longer follow-up period is required to assess the residual effects of both PRP and PRGF and comment on their long-term efficacy to reverse the natural course of the disease. Our study was a single-centered study with a smaller study group; hence, it is difficult to generalize the findings of our research concerning the population as a whole. This current study did not involve a placebo group which is always better to be included in trials comparing novel modalities such as those researched here, however, we have already compared the effects of PRGF versus a placebo agent recently and found PRGF to be much more efficacious in cases of knee osteoarthritis [32]. Further studies with a bigger sample size are required to correlate the findings of this study radiologically to qualitatively assess the regeneration of cartilage after interventions with these modalities which we plan to do soon.

From the observations made in the present study, we can conclude that intra-articular injections of PRGF are more efficacious in decreasing disease-related pain and improving joint function when compared to intra-articular injections of PRP in patients with mild-to-moderate knee osteoarthritis over a 12-month follow-up period. Both PRP and PRGF equally decrease the serum levels of biomarkers specific for cartilage degradation, Coll2-1 over a 9-month follow-up from the 3^rd consecutive intra-articular injection, each administered 4 weeks apart, suggesting that they possess the properties to halt the disease-related cartilage degradation and regenerate the affected sites of the articular surface.

This original article studies and compares the effects of intra-articular injections of platelet rich plasma and plasma rich in growth factors and quantify their effects in knee osteoarthritis by way of biomarker for cartilage degradation, Col2-1.