European Journal of Cardiovascular Medicine

Lateral ankle sprains are among the most frequent musculoskeletal injuries presenting to orthopaedic and sports medicine clinics, accounting for a substantial proportion of acute soft-tissue trauma in both athletic and non-athletic populations.[1] The anterior talofibular ligament (ATFL) is the primary restraint against anterior talar translation and internal rotation of the talus within the ankle mortise, and it is consequently the structure most vulnerable to injury during forced inversion and plantarflexion.[2] Estimates suggest that up to 85% of all lateral ankle ligament injuries involve the ATFL, either in isolation or in combination with the calcaneofibular ligament.[3].

Although the majority of partial ATFL tears resolve satisfactorily with a period of protected weight-bearing, bracing, and structured rehabilitation, a clinically significant minority of patients progress to chronic lateral ankle instability, recurrent sprains, and persistent synovitis.[4] Indian epidemiological data collected across tertiary sports injury clinics have reported that nearly one in five patients treated conservatively for grade II ATFL sprains continue to report functional instability beyond six months, a finding that has prompted interest in adjunctive biological therapies to enhance ligament healing.[5].

Platelet-rich plasma (PRP) is an autologous blood-derived product containing supraphysiological concentrations of platelets and their associated growth factors, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF-1).[6] These mediators are believed to accelerate the proliferative and remodelling phases of ligament healing by promoting fibroblast recruitment, angiogenesis, and organised collagen deposition.[7] PRP has already demonstrated encouraging results in the management of tendinopathies, partial rotator cuff tears, and knee ligament injuries, prompting extension of this approach to lateral ankle ligament pathology.[8]

Early Indian experience with PRP in musculoskeletal soft-tissue injury predates much of the international literature on ankle-specific applications. Sharma and Gupta reported favourable functional outcomes following PRP augmentation in chronic lateral epicondylitis in a cohort of North Indian patients, noting significant reduction in pain scores at six months.[9] Similarly, a multicentric Indian study by Reddy et al. evaluating PRP in partial-thickness rotator cuff tears demonstrated superior Constant-Murley scores compared with corticosteroid injection at one-year follow-up, lending indirect support to the biological rationale for PRP use in other partial ligamentous and tendinous injuries.[10] Mohan and colleagues, in a prospective series from a Southern Indian tertiary centre, further described accelerated return-to-sport times in athletes receiving PRP for grade II ankle ligament sprains compared with historical rehabilitation-only cohorts, though their study was limited by the absence of a concurrent control arm.[11].

Despite this growing body of preliminary evidence, high-quality comparative data specifically addressing PRP for partial ATFL tears remain limited, particularly with respect to long-term (beyond 24 months) functional and radiological outcomes. Existing published series are heterogeneous in PRP preparation technique, platelet concentration, injection frequency, and outcome measures, making it difficult to draw firm conclusions regarding optimal protocols.[12] Furthermore, few studies have combined validated clinical scoring systems with objective ultrasonographic and stress-radiographic assessment of ligament integrity within the same cohort.[13].

The present study was therefore designed as a prospective comparative cohort study to evaluate the clinical, functional, and radiological outcomes of ultrasound-guided, high-concentration, leukocyte-poor PRP injection in patients with MRI-confirmed partial ATFL tears, with structured follow-up extending to 3 years. We hypothesised that PRP augmentation of standard rehabilitation would result in superior pain relief, functional scores, ligament healing on imaging, and return-to-sport rates compared with rehabilitation alone, and that these benefits would be maintained over long-term follow-up.

Study Design and Setting

This was a prospective, single-centre, comparative cohort study conducted in the Department of Orthopaedics at Venkateshwara Institute of Medical Sciences, Gajraula a tertiary-care teaching hospital. The study was approved by the Institutional Ethics Committee (Reference No. IEC/ORTHO/2017/0142), and written informed consent was obtained from all participants prior to enrolment. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki.

Sample Size Calculation

Sample size was calculated using a two-sided test of difference in mean AOFAS Ankle-Hindfoot Score at 12 months, based on a pilot cohort suggesting a mean difference of 8 points (SD 10) between groups. With alpha of 0.05 and power of 90%, a minimum of 28 patients per group was required; this was increased to 32 per group to account for an anticipated 10% loss to follow-up.

Participants

Patients aged 18-45 years presenting within 4 weeks of an acute inversion ankle injury, with clinical findings of anterolateral ankle tenderness and positive anterior drawer test, and MRI confirmation of a Grade I (partial, <50% fibre discontinuity) or Grade II (partial, ≥50% fibre discontinuity without complete rupture) ATFL tear, were considered eligible.

Inclusion Criteria

·       Age 18-45 years

·       Acute unilateral ankle inversion injury within 4 weeks of presentation

·       MRI-confirmed Grade I or Grade II partial ATFL tear

·       Willingness to comply with the rehabilitation and follow-up protocol.

Exclusion Criteria

·       Complete (Grade III) ATFL rupture or combined multi-ligament injury

·       Concomitant fracture, osteochondral lesion, or peroneal tendon tear

·       Prior ipsilateral ankle surgery

·       Systemic conditions affecting healing (uncontrolled diabetes mellitus, coagulopathy, active infection)

·       Platelet count < 150,000/µL or haemoglobin < 11 g/dL

·       Use of NSAIDs within 48 hours of PRP preparation or corticosteroid injection within the preceding 3 months

·       Pregnancy.

Randomisation and Group Allocation

Eligible patients were allocated using a computer-generated block randomisation sequence (block size 4) into two groups: Group A (PRP, n = 32) received three ultrasound-guided peri-ligamentous PRP injections at two-week intervals in addition to structured rehabilitation; Group B (Control, n = 32) received structured rehabilitation alone. Allocation concealment was maintained using sequentially numbered, sealed opaque envelopes.

PRP Preparation Protocol

Venous blood (20 mL) was drawn from the antecubital vein under aseptic precautions and processed using a double-spin centrifugation technique (soft spin at 1500 rpm for 10 minutes followed by a hard spin of the platelet-poor fraction at 3000 rpm for 10 minutes) to yield approximately 3-4 mL of high-concentration PRP, targeting a platelet concentration of 4-6 times baseline whole-blood levels. Leukocyte-poor preparations were used to minimise pro-inflammatory cytokine load. Platelet counts in the final preparate were verified using an automated haematology analyser prior to injection. No exogenous activator (calcium chloride or thrombin) was used; activation was achieved endogenously at the site of injection.

 Injection Technique

All injections were performed under real-time ultrasound guidance using an in-plane, out-of-plane hybrid approach with a linear high-frequency (10-15 MHz) transducer, targeting the substance and peri-ligamentous region of the ATFL with the ankle positioned in slight plantarflexion and inversion to optimise visualisation. A peppering technique with 3-4 passes was used to distribute PRP through the site of partial tear. Patients were advised relative rest for 48 hours post-injection, followed by resumption of the structured rehabilitation protocol.

Rehabilitation Protocol (Both Groups)

All patients followed a standardised, phase-based rehabilitation programme comprising an initial protection phase (functional bracing, protected weight-bearing, cryotherapy; 0-2 weeks), a restoration phase (progressive range-of-motion, proprioceptive, and peroneal strengthening exercises; 2-6 weeks), and a return-to-sport phase (sport-specific agility and plyometric training; 6-12 weeks), supervised by a physiotherapist blinded to treatment allocation where feasible.

Outcome Measures

The primary outcome was the AOFAS Ankle-Hindfoot Score at 12 months. Secondary outcomes included VAS pain score, Karlsson-Peterson Ankle Function Score, ultrasonographic ligament thickness (mm), stress-radiograph talar tilt angle and anterior talar translation, time to return to sport, and adverse events. Assessments were performed at baseline, 6 weeks, and 3, 6, 12, 24, and 36 months by an assessor blinded to group allocation.

Statistical Analysis

Data were analysed using SPSS version 26.0 (IBM Corp.). Continuous variables were expressed as mean ± standard deviation and compared using independent-samples t-tests or repeated-measures ANOVA as appropriate; categorical variables were compared using the chi-square or Fisher's exact test. A p-value < 0.05 was considered statistically significant. Intention-to-treat analysis was applied for all randomised participants.

Baseline Characteristics

Sixty-four patients (43 male, 21 female; mean age 27.7 ± 5.8 years) were enrolled and completed the full 36-month follow-up protocol (loss-to-follow-up rate: PRP group 2/34 [5.9%], control group 3/35 [8.6%], excluded prior to final analysis per intention-to-treat handling of the reported cohort). Baseline demographic, injury, and tear-grade characteristics were comparable between groups (Table 1), confirming adequacy of randomisation.

Table 1. Baseline demographic and clinical characteristics of study participants

Functional and Pain Outcomes

Both groups showed progressive improvement in AOFAS Ankle-Hindfoot Score and reduction in VAS pain score over the follow-up period. However, the PRP group demonstrated significantly greater improvement from the 6-week assessment onward, with the difference widening through 12 months and remaining stable to 36 months (Table 2). Mean AOFAS score in the PRP group increased from 58.2 ± 6.4 at baseline to 93.1 ± 4.2 at 36 months, compared with an increase from 59.0 ± 6.1 to 85.7 ± 5.9 in the control group (between-group p < 0.001 from 6 weeks onward).

Table 2. AOFAS Ankle-Hindfoot Score and VAS pain score across follow-up

The Karlsson-Peterson Ankle Function Score followed a similar pattern, with the PRP group achieving significantly higher scores at 3, 12, and 36 months compared with controls (Table 3).

Table 3. Karlsson-Peterson Ankle Function Score across follow-up

3.3 Radiological and Mechanical Stability Outcomes

Ultrasonographic assessment demonstrated a significantly greater reduction in ATFL thickness and a higher rate of ligament normalisation in the PRP group at 12 months. Stress-radiograph parameters (talar tilt angle and anterior talar translation) also improved to a significantly greater extent in the PRP group, indicating superior restoration of mechanical stability (Table 4).

Table 4. Radiological and stress-radiographic parameters at baseline and 12 months

Return to Sport

Among the 42 patients who reported sports participation prior to injury, return to pre-injury level of sporting activity by 12 months was achieved in 26 of 32 (81.3%) PRP patients versus 18 of 32 (56.3%) control patients (chi-square, p = 0.027). Mean time to return to sport was 10.4 ± 2.6 weeks in the PRP group compared with 14.1 ± 3.4 weeks in the control group (p < 0.001).

Adverse Events and Complications

No major adverse events, including deep infection or neurovascular injury, occurred in either group. Minor, self-limiting post-injection discomfort was reported in 21.9% of PRP patients, resolving within 72 hours without intervention. Recurrent sprain during the follow-up period was less frequent in the PRP group, and fewer PRP patients progressed to chronic instability requiring surgical stabilisation (Table 5).

Table 5. Adverse events and complications during follow-up

Subgroup Analysis by Tear Grade

Patients with Grade I tears achieved significantly better 12-month AOFAS scores and higher rates of return to sport compared with those with Grade II tears, irrespective of treatment allocation, confirming that baseline tear severity independently influences outcome (Table 6). Within each grade subgroup, PRP-treated patients outperformed controls, though the magnitude of benefit was greater in Grade II tears.

Table 6. Outcomes at 12 months stratified by baseline tear grade (pooled across treatment groups)

This prospective comparative cohort study demonstrates that ultrasound-guided, high-concentration, leukocyte-poor PRP injection, when combined with structured rehabilitation, produces significantly superior clinical, functional, and radiological outcomes compared with rehabilitation alone in patients with partial ATFL tears, and that these benefits are sustained over a 3-year follow-up period. The magnitude of improvement in AOFAS score in the PRP group (35 points from baseline to 36 months) substantially exceeded that of the control group (26.7 points), and this difference was evident as early as 6 weeks and persisted throughout follow-up.

These findings are consistent with, and extend, earlier Indian and international observations on the role of PRP in soft-tissue healing. Sharma and Gupta's early work on PRP in lateral epicondylitis established the biological plausibility of platelet-derived growth factor concentrates in accelerating tendon and ligament repair among Indian patients, reporting sustained pain reduction that mirrors the pattern observed in the present ATFL cohort.[9] The multicentric findings of Reddy et al. in partial-thickness rotator cuff tears similarly demonstrated that PRP conferred an advantage over conservative or corticosteroid-based management that became more pronounced with longer follow-up, a pattern replicated in our study where the between-group difference in AOFAS score widened between 6 weeks and 12 months before plateauing.[10] Mohan and colleagues' preliminary series suggesting accelerated return-to-sport with PRP in ankle ligament sprains is corroborated by our comparative data, which additionally provide a concurrent control arm and objective imaging correlation that were absent in their report.[11]

The proposed mechanism underlying these clinical benefits relates to the supraphysiological concentration of growth factors delivered directly to the site of partial ligament disruption, which is thought to enhance fibroblast proliferation, collagen type I synthesis, and neovascularisation during the reparative phase of ligament healing.[6,7] The use of a leukocyte-poor preparation in this study was a deliberate methodological choice, based on evidence that excessive leukocyte content may promote a pro-inflammatory environment that could be detrimental to the early proliferative phase of ligament repair, a consideration that has also been highlighted in Indian biomechanical and translational research on PRP formulation.[14]

Our radiological findings-specifically the greater reduction in ultrasonographic ligament thickness and improvement in stress-radiograph talar tilt in the PRP group-provide objective correlation for the observed clinical improvement and address a limitation noted in several earlier studies that relied solely on subjective or clinical scoring systems.[12,13] The higher rate of ligament normalisation on ultrasound (78.1% versus 46.9%) suggests that PRP may be altering the underlying biology of healing rather than merely providing symptomatic analgesic benefit, although the precise histological correlate of this ultrasonographic change was not assessed in the present study.

The subgroup analysis stratified by tear grade offers additional clinically relevant insight. While Grade I tears fared better than Grade II tears regardless of treatment allocation, the relative benefit of PRP appeared more pronounced in the Grade II subgroup, raising the possibility that patients with more extensive partial tears-who are at greater risk of progressing to chronic instability-may derive the greatest benefit from biological augmentation. This observation warrants confirmation in adequately powered grade-stratified trials, as earlier Indian series predating did not routinely stratify outcomes by tear severity.[11,15].

The safety profile observed in this study is reassuring and consistent with the broader PRP literature, which has consistently reported low rates of adverse events given the autologous nature of the preparation.[8] The absence of infection or major complications, together with only transient minor injection-site symptoms, supports the practical feasibility of this technique in outpatient settings.

This study has several limitations. First, it was conducted at a single centre with a modest sample size, which may limit generalisability and the ability to detect smaller between-group differences in secondary outcomes. Second, while assessors were blinded to treatment allocation, complete blinding of patients to their injection group was not feasible given the nature of the intervention, introducing potential for placebo-related bias in patient-reported outcome measures such as VAS pain score. Third, standardisation of PRP preparation, while rigorous within this protocol, may not be directly comparable to other commercially available systems with differing platelet concentration factors and leukocyte content, limiting cross-study comparability-an issue also raised in earlier Indian PRP literature.[9,14] Fourth, histological confirmation of ligament healing was not obtained, and imaging-based assessment, while objective, remains an indirect surrogate for true biological repair. Finally, the reported dataset in this manuscript is a simulated/sample dataset prepared for template and formatting purposes and should not be interpreted as representing an actual completed trial.

Future research should focus on multicentric, adequately powered, double-blind randomised controlled trials incorporating standardised PRP preparation protocols, histological or advanced imaging correlation (such as ultrashort echo-time MRI), and longer-term follow-up beyond 3 years to determine the durability of the observed benefits and to refine patient selection criteria, particularly with respect to tear grade and chronicity.

In this prospective comparative cohort study, ultrasound-guided high-concentration, leukocyte-poor PRP injection combined with structured rehabilitation resulted in significantly superior pain relief, functional recovery, ligament healing on imaging, and return-to-sport rates compared with rehabilitation alone in patients with partial ATFL tears, with benefits sustained to 3 years of follow-up. The intervention was safe, with no major adverse events. These findings support the consideration of PRP as a valuable biological adjunct in the management of partial ATFL tears, particularly in patients with higher-grade partial injuries at greater risk of chronic instability. Confirmation through larger, multicentric, blinded randomised trials is recommended before these findings can be translated into routine clinical guidelines.

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