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1 3 Knee Surg Sports Traumatol Arthrosc DOI 10.1007/s00167-014-3260-6 KNEE Hamstrings anterior cruciate ligament reconstruction with and without platelet rich fibrin matrix M. Del Torto · D. Enea · N. Panfoli · G. Filardo · N. Pace · M. Chiusaroli Received: 29 August 2013 / Accepted: 20 August 2014 © European Society of Sports Traumatology, Knee Surgery, Arthroscopy (ESSKA) 2014 Conclusions The procedure described for PRFM aug- mentation in ACL STG reconstruction does not improve radiologic graft integration and knee stability after 1 year and should not be used by clinicians to this purpose. How- ever, it may result in a short-term improvement of patient- reported knee function, and future research should focus on further developing PRP treatment to optimize ACL clinical outcome. Level of evidence III. Keywords PRFM · ACL · Semitendinosus-gracilis · MRI · Platelet-derived growth factors Introduction A large number of anterior cruciate ligament (ACL) repair techniques have been described, but the success rate in terms of mid-term clinical outcome still does not exceed 85–90 % [8, 20]. Among the most commonly used proce- dures autologous bone–patellar tendon–bone graft offers the strongest healing potential because it relies mainly on bone-to-bone integration between graft bone plug and tun- nel wall [27, 30]. However, this technique has some donor- site morbidity that led for the search of alternative graft sources or additive biological treatments on the donor site [6, 35, 46]. Autologous hamstring grafts have less donor- site morbidity but, regarding bone integration, rely solely on tendon-to-bone healing. This process occurs slowly leading to concerns about graft pullout and slippage result- ing in joint instability and potential failure of the graft [35]. Therefore, integration of semitendinosus–gracilis (STG) graft with the bone tunnel is a fundamental aspect to achieve in the postoperative phase, and both surgeons in the operating room and researchers in the laboratory are Abstract Purpose Anterior cruciate ligament (ACL) rupture is the most common complete ligamentous injury in the knee. Many studies explored ACL graft integration and matu- ration, but only a few assessed the application of platelet rich fibrin matrix (PRFM) as augmentation for ACL recon- struction. The main aim of this study was to test the PRFM augmentation in terms of graft–bone integration and knee stability. The secondary aim was to investigate patient- reported functional status. Methods Prospective evaluation has been done in two consecutive series of patients who underwent ACL recon- struction with semitendinosus and gracilis (STG) grafts: 14 patients were operated with PRFM augmentation and 14 patients without PRFM augmentation. Objective clinical evaluation (Rolimeter) and MRI evaluation were performed at 1 year from surgery. Subjective evaluation (IKDC) was performed pre-operatively and at 6 months, 1 and 2 years from surgery. Results A statistically significant difference was not detected between the two groups in terms of MRI and objective clinical evaluation, although PRFM-augmented patients showed a statistically significant higher clinical improvement. M. Del Torto (*) · N. Panfoli · N. Pace · M. Chiusaroli Department of Orthopaedics and Traumatology, Jesi Civil Hospital, Viale Della Vittoria, 76, Jesi, AN, Italy e-mail: [email protected] D. Enea Department of Orthopaedics, Polytechnic University of Marche, Ancona, Italy G. Filardo II Clinic - Biomechanics Laboratory, Rizzoli Orthopaedic Institute, Bologna, Italy

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Page 1: Hamstrings anterior cruciate ligament reconstruction with and without platelet rich fibrin matrix

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Knee Surg Sports Traumatol ArthroscDOI 10.1007/s00167-014-3260-6

Knee

Hamstrings anterior cruciate ligament reconstruction with and without platelet rich fibrin matrix

M. Del Torto · D. Enea · N. Panfoli · G. Filardo · N. Pace · M. Chiusaroli

Received: 29 August 2013 / Accepted: 20 August 2014 © european Society of Sports Traumatology, Knee Surgery, Arthroscopy (eSSKA) 2014

Conclusions The procedure described for PRFM aug-mentation in ACL STG reconstruction does not improve radiologic graft integration and knee stability after 1 year and should not be used by clinicians to this purpose. How-ever, it may result in a short-term improvement of patient-reported knee function, and future research should focus on further developing PRP treatment to optimize ACL clinical outcome.Level of evidence III.

Keywords PRFM · ACL · Semitendinosus-gracilis · MRI · Platelet-derived growth factors

Introduction

A large number of anterior cruciate ligament (ACL) repair techniques have been described, but the success rate in terms of mid-term clinical outcome still does not exceed 85–90 % [8, 20]. Among the most commonly used proce-dures autologous bone–patellar tendon–bone graft offers the strongest healing potential because it relies mainly on bone-to-bone integration between graft bone plug and tun-nel wall [27, 30]. However, this technique has some donor-site morbidity that led for the search of alternative graft sources or additive biological treatments on the donor site [6, 35, 46]. Autologous hamstring grafts have less donor-site morbidity but, regarding bone integration, rely solely on tendon-to-bone healing. This process occurs slowly leading to concerns about graft pullout and slippage result-ing in joint instability and potential failure of the graft [35].

Therefore, integration of semitendinosus–gracilis (STG) graft with the bone tunnel is a fundamental aspect to achieve in the postoperative phase, and both surgeons in the operating room and researchers in the laboratory are

Abstract Purpose Anterior cruciate ligament (ACL) rupture is the most common complete ligamentous injury in the knee. Many studies explored ACL graft integration and matu-ration, but only a few assessed the application of platelet rich fibrin matrix (PRFM) as augmentation for ACL recon-struction. The main aim of this study was to test the PRFM augmentation in terms of graft–bone integration and knee stability. The secondary aim was to investigate patient-reported functional status.Methods Prospective evaluation has been done in two consecutive series of patients who underwent ACL recon-struction with semitendinosus and gracilis (STG) grafts: 14 patients were operated with PRFM augmentation and 14 patients without PRFM augmentation. Objective clinical evaluation (Rolimeter) and MRI evaluation were performed at 1 year from surgery. Subjective evaluation (IKDC) was performed pre-operatively and at 6 months, 1 and 2 years from surgery.Results A statistically significant difference was not detected between the two groups in terms of MRI and objective clinical evaluation, although PRFM-augmented patients showed a statistically significant higher clinical improvement.

M. Del Torto (*) · n. Panfoli · n. Pace · M. Chiusaroli Department of Orthopaedics and Traumatology, Jesi Civil Hospital, Viale Della Vittoria, 76, Jesi, An, Italye-mail: [email protected]

D. enea Department of Orthopaedics, Polytechnic University of Marche, Ancona, Italy

G. Filardo II Clinic - Biomechanics Laboratory, Rizzoli Orthopaedic Institute, Bologna, Italy

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focused on the development of strategies to accelerate and improve the integration process of the tendon graft during ACL reconstruction [46]. While many studies in the litera-ture assessed the spontaneous integration and maturation of STG tendon graft [17, 33], less explored is the use of platelet rich plasma (PRP) as augmentation procedure to improve ACL reconstruction [41].

nowadays, it is well recognized that growth factors (GFs) play a central role in the tissue healing processes [2, 10, 15], which led to significant research efforts to clarify the potential of platelet-derived GFs to favor tissue regener-ation in tissues with low healing potential [3, 7, 13, 29, 39]. Platelet concentrates have been proposed as easy and safe solution to apply several GFs in physiologic proportions in the clinical practice [18].

The potential benefit of using such products to acceler-ate and improve ACL graft healing has been suggested by preclinical studies [22–24], while clinical studies focus-ing on remodeling and integration of the graft with the intra-operative addition of PRP have shown conflict-ing results so far [1, 19, 34]. Thus, we performed a pilot study to explore the usefulness of platelet rich fibrin matrix (PRFM) as augmentation procedure for ACL reconstruc-tion, focusing on the evaluation of both clinical and graft integration improvement. If PRP would prove to be useful, this could be of clinical relevance providing new insights in the potential of this biological approach to improve tis-sue healing and accelerate and increase the results obtained by ACL reconstruction. In vitro studies have been previ-ously reported, which assessed the presence of a substantial amount of growth factors (such as TGF-β1, PDGF, VeGF) in the PRFM applied in the present study [14]. In vivo stud-ies on rats have shown the effects of such PRFM on soft tissues, indicating that it may be beneficial for increasing neovascularization and tissue proliferation [4]. Moreover, in vivo studies on dogs have also shown a faster bone heal-ing when PRFM was used [36]. MRI imaging was chosen since it is the only non-operative way to detect graft inte-gration and ligamentization [9, 33, 38, 45].

The main aim of the present study was to test whether the addition of PRFM to the ACL graft may improve the healing in the first year through the analysis of MRI-detected bone–graft integration and objective knee laxity. Secondary aim is to document subjective knee function changes up to two years of follow-up.

Materials and methods

Two series of consecutive patients underwent to STG graft arthroscopic ACL reconstruction. The patients (n = 28, 25 men and 3 women, 17 right knees and 11 left knees) were operated by the senior surgeon (M.C.) and underwent the

same rehabilitation protocol. From the beginning of the study, the first 14 patients coming to our institution with an ACL tear and the appropriate inclusion criteria were oper-ated on with PRFM addiction (Group 1) and the following 14 patients were treated without PRFM addiction (Group 2). The treatment was not a choice of the surgeon in each case. An appropriate informed consent was obtained from all the patients before surgery.

Inclusion criteria were as follows: age between 18 and 45 years, ACL acute primary total tear, surgery at least 60 days after trauma, uninjured contralateral knee and patient acceptance with apposite informed consent. exclu-sion criteria were as follows: use of other grafts, different surgical technique, multi-ligament lesions, inflammatory/infective knee diseases, chondral lesion graded Outerbridge II or more in any knee compartment and previous surgery on the same knee.

Two Group 1 patients left voluntarily the follow-up. One Group 2 patient was not evaluated because he was not traceable and another patient was not evaluated because he refused to perform the MRI scan. Twelve patients were therefore available for both Group 1 and Group 2, which were homogeneous for the baseline characteristics.

PRFM preparation

At the time of the procedure, PRFM was prepared using Cascade Medical enterprises 2 tube kit (Cascade Medi-cal enterprises, Wayne, nJ). The blood necessary for the PRFM preparation was obtained by a venous blood har-vest using a 10-mL syringe. The collected blood was pro-cessed as for manufacturers’ instruction. Briefly, the blood was placed in citrate phosphonate dextrose in ratio 1/5 ml. The sample was initially centrifuged in the first separation tube, at 1,100 rpm for 6 min. The supernatant was then transferred to the second tube, containing calcium chloride, and spun at 1,450 rpm for 15 min. The resultant PRFM is a dense and suturable matrix containing a twofold platelet concentration with respect to peripheral blood [4].

Surgery

each patient was operated on by the senior surgeon (M.C.). The STG autologous graft was fixed in the femoral tunnel through the RIGIDFIX® system (DePuy) and in the tibial tunnel through the Bio-InTRAFIX™ system (DePuy). Fourteen patients (Group 1) were treated with PRFM, while the other 14 were considered as control group and were treated without PRFM (Group 2). When necessary, a first arthroscopic step was performed to observe and con-firm the ACL total tear.

A longitudinal skin incision above the hamstrings inser-tion was performed. Gracilis and semitendinosus tendon

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(STG) were isolated and harvested with a smooth tendon stripper. Tendons were duplicated and tied with continuous absorbable suture and simple non-absorbable suture only in the loop side. every free end was prepared with roman san-dal suture, and wires were tied like a loop in every tendon. In Group 1, the PRFM clot was sutured in the proximal STG loop and it reaches the proximal tunnel once the graft is pulled in place (Fig. 1).

Graft was afterward pre-tensioned over the workstation. When necessary meniscal lesions were treated arthroscopi-cally, intercondylic notch plasty was always performed in order to host the tendon graft and avoid impingement. Tib-ial tunnel was drilled at 50° in sagittal plane and at 25°–30° in frontal plane, positioning a guide K-wire 7 mm anterior to the posterior cruciate ligament, balancing every time this distance with the landmarks of the medial tibial spine and of the posterior side of the external meniscus. A femoral 30-mm tunnel was drilled transtibial with inconstant offset, depending on graft size and having care to obtain at least 2 mm of posterior wall. An orthogonal femoral fixation was obtained through two 3.2-mm poly(L-lactide) pins, part of Rigid-Fix® system, using the provided “U” guide. With a traction wire, the graft was positioned and fixed in the fem-oral tunnel. Distally, once the Bio-Intrafix™ tension device was positioned, the PRFM clot was inserted between the four strand of the G-ST graft before the interference screw system was applied. When the screw is forced in the clot is pushed against the graft strands and against the tunnel wall. Graft was fixed with an absorbable screw with the knee at 20° of flexion. A negative pressure intra-articular drain was positioned and left overnight.

Rehabilitation protocol

each group underwent the same rehabilitative proto-col. Passive mobilization exercises were started on first

postoperative day. Knee was protected for 30 days with a knee brace, with a progressive range of motion increase. Load bearing was allowed since day one with brace and crutches. Closed kinetics chain exercises were progres-sively introduced. The open kinetic chain exercises began after 2 months in order to improve extensor strength (leg-extension). Isokinetic rehabilitation was reserved to the end stage, before return to sport activity. Rehabilitation in water was allowed in the third week after surgery (specific exer-cises in first month and then swim, excluded breast stroke). Proprioceptive exercises were started in the second month, running was allowed after 3–4 months and unrestricted sports activity after 6 months.

Clinical evaluation

At 6 months, 1 and 2 years after surgery a clinical subjec-tive evaluation was blindly performed through the IKDC subjective score. At 1 year, a blinded objective evaluation of every patient was performed through the IKDC objective score and objective data on knee laxity were obtained with a Rolimeter arthrometer measured in mm (DJO Global, Vista, CA). Any postoperative complication was registered during the follow-up.

MRI evaluation

MRI scans of the operated knee were achieved 1 year post-operatively (esaote Artoscan, Indianapolis, In). Views per-formed were as follows: axial (turbo spin echo T1), coronal (spin echo T1, gradient echo T2 e STIR) and sagittal (spin echo T1, gradient echo T2 e STIR). The examiner was not aware of patient clinic developments (Rolimeter and IKDC) and treatment group.

A scoring scale was used to facilitate graft evaluation. Parameters considered were as follows: signal intensity

Fig. 1 a Platelet clot prepared. b, c The clot included in proximal loop preparation and sutured with roman sandal mode

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(>50 % of graft surface) and presence of synovial fluid at the tunnel–graft interface. The two evaluated parameters and the score have been validated in different previous studies [26, 42] (Table 1).

To define the signal intensity of the graft, they were compared with the intensities of the native semimembrano-sus muscle tendons in T2 sequences. They were classified as hypointense (lower intensity than that of the semimem-branosus tendon), isointense or hyperintense.

To determine the presence of synovial fluid in the femoral and tibial tunnel–graft interface, fluid-sensitive sequences (T2 potentiated) were used. The presence of syn-ovial fluid (higher signal intensity in tunnel–graft interface) was classified as positive, the absence of fluid was classi-fied as negative (Table 2).

A score was assigned to each parameter. Synovial fluid at the graft–tunnel interface: 1 point (positive), 2 points (negative). Signal intensity: 1 point (hyperintense), 2 points (isointense), 3 points (hypointense) (Table 1). The addition of both parameters allowed us to obtain an overall classifi-cation at the MRI characterization as poor integration (1–2 points) or adequate integration (3–5 points) [11, 42].

Our institutional review board approved the study: eth-ics committee (Comitato etico dell’Ospedale di Iesi, ASUR MARCHe) approved on the January 2, 2007/n49643329.

Statistical analysis

All continuous data were expressed in terms of the mean and the standard deviation of the mean. Median and 25th

and 75th percentiles were used for ordinal scores. Because of the small sizes of the groups, the Mann–Whitney test, evaluated by the exact method for small samples, was used, instead of one way AnOVA, to assess the means or the medians differences between the two groups. GLM-repeated measures with Sidak test for multiple comparisons were performed to assess differences of the scores at differ-ent follow-up times. For all tests, p < 0.05 was considered significant. Statistical analysis was carried out by means of the IBM Statistical Package for the Social Sciences (SPSS) software version 19.0.

Results

MRI results

The most useful images for comparison purposes were those corresponding to sagittal views. MRI evaluation con-sidering graft–tunnel interface and graft signal intensity provided similar results between the two examined groups (Fig. 2). In the majority of the cases, we observed a good signal quality of the graft and a scarce film of synovial fluid at the graft–tunnel interface. Both the signal intensity (liga-mentization—25 percentile, median, and 75 percentile: 2, 2, 2 vs. 2, 2, 2.5 for Group 1 and Group 2, respectively) and the fluid at the graft–tunnel interface (integration—25 per-centile, median, and 75 percentile: 1, 2, 2 for both groups) resulted similar between the two groups. The addition of the two parameters also led to similar good results in both groups (Table 2). In both groups, no patient obtained a poor score.

Clinical results

no patient had traumatic events during the follow-up. early postoperative complications were not observed. Two cases of infrapatellar hypoesthesia were observed as postopera-tive late complications.

Objective clinical evaluation both through IKDC score (25 percentile, median, and 75 percentile: 2, 3, 3 for both groups) and with Rolimeter arthromether (25 percentile, median, and 75 percentile: 1, 1, 1.5 versus 1, 1, 2 for Group 1 and Group 2, respectively) did not show any difference between the two groups (Tables 3, 4).

Subjective clinical evaluation through IKDC score showed an improvement over time in both groups. Group 1 passed from 54.3 ± 4.4 preoperatively to 90.3 ± 5.6 at 6 months (p < 0.005), 94.1 ± 5.3 at 12 months (p = 0.003), and 94.1 ± 4.7 at 2 years of follow-up (12 vs. 24 n.s.). Group 2 passed from 51.6 ± 7.0 preopera-tively to 82.5 ± 13.7 at 6 months (p < 0.005), 84.1 ± 14.2 at 12 months (p = 0.007), and 85.4 ± 12.2 at 2 years of

Table 1 MRI evaluation according to Figueroa et al. [11]

Score (points)

Integration: synovial fluid at tunnel–graft interface (femoral or tibial)

Positive 1

negative 2

Ligamentization: graft signal pattern (>50 %)

Hypointense 3

Isointense 2

Hyperintense 1

Characterization of graft

Poor 2

Adequate 3–5

Table 2 MRI evaluation at 12 months of follow-up

MRI evaluation

Group 1 (n = 12) 3.6 ± 0.5

Group 2 (n = 12) 3.7 ± 0.8

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follow-up (12 vs. 24 n.s.). The comparison of the subjec-tive scores showed similar basal values but statistically sig-nificant better results for Group 1 at 12 (p = 0.028) and 24 months (p = 0.032). To confirm the different outcome observed in the absolute score values, we also analyzed the percentage of improvement with respect to the basal

condition of every patient at all follow-ups, demonstrating again that Group 1 obtained a significantly higher subjec-tive improvement at 12 months (p = 0.029) and 24 months (p = 0.034) (Table 5).

Discussion

The most important findings of the present study were that the addition of PRFM into bone tunnels during ACL recon-struction procedure did not lead to an improved knee stabil-ity or MRI appearance of graft integration and maturation. On the other side, a clinical improvement up to 24 months was observed in PRFM-treated patients.

A key aspect to guarantee durable stability outcomes when performing an ACL reconstruction with STG grafts is to achieve optimal bone–graft integration [25, 31]. Most

Fig. 2 a example of T1 image of a graft with a good signal quality (isointense compared to semimembranosus). b example of T1 image of a graft with poor signal quality (hyperintense compared to semimembrano-sus). c T1 image with presence of synovial fluid at the femoral graft–tunnel interface. d T1 image with absence of synovial fluid at the femoral graft–tunnel interface

Table 3 IKDC objective modulus at 12 months postoperatively

evaluation feature Group 1 Group 2

effusion 12 A (100 %) 12 A

Passive motion deficit 12 A 12 A

Ligament examination 8A 7A

2B 5B

2C –

Table 4 Objective Lachman test with Rolimeter 12 month postopera-tively

Group 1 Group 2

1–2 mm 9 8

3–5 mm 3 4

6–10 mm – –

>10 mm – –

Table 5 IKDC subjective modulus

* Statistical significant

6 months 12 months 24 months

Group 1 (n = 12) 69.6 ± 10.5* 74.9 ± 4.1* 78.3 ± 5.3*

Group 2 (n = 12) 62. 6 ± 10.5 63.4 ± 10.6 65.1 ± 9.8

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studies have shown that the ligamentization phase, where the graft’s structural and biomechanical characteristics may become similar to those of an intact ACL, with the develop-ment of Sharpey fibers that show integration with the bone tunnels lasts at least 4 to 6 months. It has not been pos-sible to define the end of this phase because some changes occur even years after the reconstruction and, currently, this aspect is still a matter of discussion [9, 31, 32].

The possibility to accelerate and improve the integration process and therefore also knee stability is highly desirable. nowadays, it is extensively recognized that platelet-derived GFs play a central role in tissue’s healing and regeneration processes [2, 7, 10, 15, 18, 39]. The specific amount of GFs (such as TGF-β1, PDGF, VeGF) contained in the PRFM adopted in this study has already been shown and has been proved to be comparable to that of other commercially available systems [4]. The effect of such PRFM on soft tissues proliferation and neovascularization has also been shown [14]. Moreover, in vivo studies on dogs have also shown an improved bone healing when PRFM was adopted to enhance the bone filling of extraction socket [36].

A few studies suggest the usefulness of platelet products to favor ACL reconstruction [41]. Among these, some were more focused on the enhancement of the ligamentization process. Ventura et al. [42] performed ACL reconstruction with (n = 10) or without (n = 10) a platelet concentrate, which was placed in both tunnels directly with autologous thrombin: at 6 months there were no significant differences concerning the clinical examination. However, the CT scan highlighted a significant improvement in the ACL density in the platelet-treated group with respect to the untreated group which showed a more heterogeneous nature. It is worthy to mention that these authors also reported an adverse event in the PRP augmented group one patient had a synovitic reaction: the new ACL was increased in size, hypertrophic and surrounded by soft tissue reaction. Orrego et al. [26] found 100 % grafts with intensity similar to native posterior cruciate ligament in the platelet group but only 78 % in the control group at 6 months. Figueroa et al. [11] evaluated the grafts at 6 months postoperatively and found 63.2 % hypointense grafts in the platelet group but only 42.1 % hypointense grafts in the control group. The difference was not statistically significant but the trend suggested a better ligamentization of the graft in the treated group. nin et al. [25] confirmed these findings at 24 months documenting a 21 % lower intensity on proton density-weighted magnetic resonance images and 23 % lower intensity on T2-weighted magnetic resonance images in the platelet group. In an MRI study, Radice et al. [31] focused on the time course of graft signal and demonstrated a 48 % reduction in the time required to achieve a complete homogeneous graft signal when PRP was used for ACL graft augmentation (from 369 to 177 days on average).

Sánchez et al. [32] assessed ACL grafts in terms of gross morphology and histologically. Arthroscopic gross evalua-tion showed excellent ratings for 57.1 % of platelet-treated ACL grafts, but only 33.3 % in the control group, and his-tologic assessment showed a significantly better maturity index and more newly developed synovial tissue envelop-ing in the platelet-treated grafts.

Whereas these articles almost consistently show an improvement in the graft ligamentization process, less documented and more controversial is the role of PRP in another key aspect for the success of ACL reconstruction: the integration of the graft. Orrego et al. [26] showed no significant effect of the platelet concentrate on the osteo-ligamentous interface or tunnel widening evolution at 6 months. Vadala’ et al. [40] evaluated the efficacy of PRP in reducing femoral and tibial tunnel enlargement at 10–16 months but failed to prove any benefit from this aug-mentation strategy, which according to Figueroa et al. [11] could even lead to a lower integration, as suggested by the finding of a greater number of cases positive for the pres-ence of synovial fluid at the tunnel–graft interface. Oppo-sitely, a study of Vogrin et al. [44] focused on the MRI analysis of graft vascularization in PRP-treated and control groups. no differences of the intra-articular portion of ACL grafts was found at 12 weeks, but the revascularization was enhanced in the interface zone between the graft and the bone tibial tunnel, and the authors showed that ham-string grafts augmented by platelet-derived GFs had a sig-nificantly better anteroposterior knee stability at 6 months [43].

One of the greatest weaknesses of the previous studies on ACL graft–bone integration is that MRI was performed earlier than 1 year postoperatively, which has been shown to be the minimum time needed to complete graft integra-tion [11]. Therefore, we performed a 1-year MRI evalua-tion of bone–graft integration and graft maturation. Another strength of our study is clinical follow-up performed up to 2 years postoperatively while the previous studies per-formed a shorter follow-up [11, 31, 41].

Our study did not show any statistically significant dif-ferences between the PRFM-augmented group and the control group. Similarly, the objective clinical evaluation (Rolimeter) performed at 1 year did not show any differ-ence among the two groups. However, our study obtained another interesting finding: a statistically significant improvement in the clinical outcome was obtained after PRP augmentation in terms of subjective function. The improvement was not limited to the postoperative phase but extended to the 2-year follow-up. The stability over time of this effect suggests that it may not just be a pain modulation mediated by platelets, but that it is likely that PRP might play a role in the healing mechanisms. It is rec-ognized that PRP injections can reduce pain and improve

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knee function and quality of life with short-term efficacy [5, 12, 16, 21, 28, 37]. The trauma causing ACL rupture can be also responsible for an insult to other tissues and there-fore an altered joint homeostasis, which is further affected by the surgical trauma itself. We could speculate that PRP can act favoring a better joint homeostasis both directly, through the release of GFs and bioactive molecules, and indirectly limiting the intra-articular bleeding from the tun-nels. However, these are just hypothesis, since the design of this study does not allow to demonstrate the mechanism of action of PRP.

This study has some limitations. There is the lack of a randomization; however, two consecutive series of patients were compared, the treatment was not a choice of the sur-geon in each case, and the outcome was blindly evaluated.

The number of treated patients in each group was small. A larger group size was originally planned according to the evaluation of Figueroa et al. [11]. However, even though 14 patients in each group are clearly a small number, the uncertain findings of the current literature prompted us to perform an intermediate evaluation to check for the useful-ness of this approach with respect to the primary outcome of this study (graft integration and knee stability). This pilot evaluation demonstrated too similar results, in terms of graft integration and knee stability among PRFM-treated and control patients at the examined time points, in order to justify the treatment of other patients with this procedure.

Another weakness of this study is timing of evalua-tion, with an MRI evaluation only at 12 months. The MRI characteristics of STG ACL grafts have been previously described, with complete maturation by 18–24 months after surgery, but most of the study attention is focused on the first phases of the maturation process at follow-up lesser than 12 months, which are of major importance for the return of patients to previous sport activity level. even though no MRI difference has been shown at 12 months postoperatively among PRP-treated and control group in terms of bone–graft integration [11], an earlier MRI evalua-tion at 3 and/or 6 months should have been desirable in the present study. Similarly, an earlier clinical and Rolimeter evaluation could have been performed to detect an earlier difference among groups.

The findings of the present study prompted us not to continue with this clinical application of PRFM. In fact, from the data obtained, it resulted very unlikely that a dif-ference among groups (PRFM-treated and controls) could be seen even doubling the group size.

However, despite the small number of patients, it was possible to demonstrate another effect of PRFM augmen-tation: besides the lack of complications, we also docu-mented a significant subjective clinical improvement up to 2 years of follow-up.

These results suggest that the use of PRFM can be bene-ficial in ACL reconstruction, but that the application modal-ity adopted in the present study may not be the optimal. It can be hypothesized that the clinical benefit of PRFM was due to the restoration of a better joint homeostasis or to the stimulation of intra-articular tissues anabolism. Therefore, an easier strategy such as the injective procedure should be investigated, instead of the more cumbersome and time-consuming intra-tendinous suture.

Conclusions

A key aspect to guarantee durable stability outcomes when performing an ACL reconstruction with STG grafts is to achieve optimal bone–graft integration. Thus, platelet-derived GFs augmentation was used aiming to accelerate and improve the healing processes. However, the results of this study suggested that that the application of PRFM in femoral and tibial tunnels does not provide a significant graft integration and knee stability at 12 months postop-eratively, while it can result in an improvement of patient-reported knee function at 12 and 24 months postoperatively. The real potential and the most appropriate application modalities of blood derivatives for ACL surgery have to be further evaluated by future specifically designed studies.

Conflict of interest The authors declare that they have no conflict of interest.

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