To systematically review the currently available literature concerning the application of biologic agents such as platelet-rich plasma (PRP) and stem cells to promote anterior cruciate ligament (ACL) healing.

A systematic review of the literature was performed on the use of biologic agents (i.e., PRP or stem cells) to favor ACL healing during reconstruction or repair. The following inclusion criteria for relevant articles were used: Clinical reports of any level of evidence, written in English language, on the use of PRP or stem cells during ACL reconstruction/repair. Exclusion criteria were articles written in other languages, reviews, or studies analyzing other applications of PRP/stem cells in knee surgery not related to promoting ACL healing.

The database search identified 394 records that were screened. A total of 23 studies were included in the final analysis: In one paper stem cells were applied for ACL healing, in one paper there was a concomitant application of PRP and stem cells, whereas in the remaining 21 papers PRP was used. Based on the ACL injury pattern, two papers investigated biologic agents in ACL partial tears whereas 21 papers in ACL reconstruction. Looking at the quality of the available literature, 17 out of 21 studies dealing with ACL reconstruction were randomized controlled trials. Both studies on ACL repair were case series.

There is a paucity of clinical trials investigating the role of stem cells in promoting ACL healing both in case of partial and complete tears. The role of PRP is still controversial and the only advantage emerging from the literature is related to a better graft maturation over time, without documenting beneficial effects in terms of clinical outcome, bone-graft integration and prevention of bony tunnel enlargement.

Anterior cruciate ligament (ACL) injuries are among the most common conditions treated in everyday orthopaedic practice, with an increasing incidence in the past years due to a concurrent increase of sports activities among the general population[1,2]. ACL lesions can be distinguished in complete tears, generally treated by reconstruction, and partial tears (i.e., incomplete tears of one or both ACL bundles). Their management can be very challenging, ranging from non-operative treatment to surgery (augmentation or traditional reconstruction), depending on the patients’ symptoms and functional needs[3]. Epidemiological studies reveal an average incidence of 30 ACL injuries per 100000 people annually. Every year 175000 patients undergo ACL reconstruction in the United States[4]. These numbers highlight the social and economical impact of ACL injuries, and therefore justify the large interest in optimizing the treatment strategies for this particular injury. Several reconstructive techniques have been proposed in the past decades, mainly differing in terms of graft selection and graft fixation. The overall results are quite satisfactory, even at long-term evaluation, without a difference in terms of outcome among different surgical techniques[5,6]. Nevertheless, a documented failure rate of up to 14% for ACL reconstruction[7], stimulates scientific efforts to find solutions that could promote better graft maturation and healing to minimize the risk of failure and to allow a faster recovery for patients. Beyond maximizing the results of ACL reconstruction, there is an increasing demand for minimally invasive options to enhance intrinsic ACL healing in case of partial ruptures. These injuries represent a substantial amount of ACL injuries, whose treatment algorithm is still controversial. The possibility of promoting ACL healing without reconstruction is regarded as an attractive perspective, due to the inherent lower surgical morbidity and the faster return to physical activities.

The recent progress in the field of regenerative medicine has led to the application of biologic agents (platelet-derived growth factors and stem cells), which could provide a positive stimulus to tissue healing. Platelet-rich plasma (PRP) is currently the most exploited biological augmentation used in orthopaedic practice, both for the treatment of degenerative disease (like osteoarthritis and tendinopathies), and sports-related injuries[8,9]. It is an autologous blood derived product which is obtained by centrifugation or filtration of peripheral blood in order to concentrate platelets, which are a reservoir of several growth factors and bioactive molecules involved in tissue homeostasis and anabolism[10,11]. Several in vitro and animal studies demonstrated that intra-ligamentary administration of PRP determines an increase in cellular density and neovascularization of the ACL. This results in a better organization of collagen fibers for superior tensile resistance and biomechanical properties[12-14]. In light of these promising pre-clinical data, and also due to the easy preparation modalities, platelet-rich products are used more and more by clinicians from all over the world. In more recent times, mesenchymal stem cells from different sources have been proposed to augment ACL reconstruction or repair. In this case, flourishing pre-clinical literature suggests that stem cell administration could stimulate tissue maturation, improve histological appearance, and favor bone-to-tendon integration[15,16]. Therefore, the implementation of experimental techniques in the clinical practice seems reasonable. A relevant number of clinical reports have been published investigating the actions of these fashionable biological strategies so far. The aim of the present paper is to systematically review the available literature concerning the application of PRP or stem cells to stimulate ACL healing, and to trace the state of the art in the use of biologic agents in this particular field of sports medicine. Moreover, this paper reveals if these novel strategies could really play a beneficial role in promoting graft maturation and healing and provide a better clinical outcome in the treatment of complete and partial ACL lesions.

A systematic review of the literature was performed on the use of biologic agents (i.e., PRP or stem cells) to promote ACL healing during surgical reconstruction or repair. The search was conducted on the PubMed database on March 31, 2016 using the following formula: [(PRP or platelet concentrate or platelet gel or platelet rich plasma or ACP or autologous conditioned plasma or PRGF or PRF or platelet lysate or platelet derived growth factors or plasma growth factors or platelet rich fibrin) or (stem cell or mesenchymal or mesenchymal stem cell or bone marrow concentrate or bone marrow aspirate or adipose derived or peripheral blood)] and (ACL or anterior cruciate ligament).

Screening process and analysis were conducted separately by two independent observers (Di Matteo B and Andriolo L). First, the articles were screened by title and abstract. The following inclusion criteria for relevant articles were used during the initial screening of titles and abstracts: Clinical reports of any level of evidence, written in English language, published in the past 20 years (1996-2016), on the use of PRP or stem cells during ACL reconstruction/repair. Exclusion criteria were articles written in other languages, reviews, or studies analyzing other applications of PRP/stem cells in knee surgery not related to promoting ACL healing. In the second step, the full texts of the selected articles were screened, with further exclusions according to the previously described criteria. Moreover, the articles not reporting clinical, magnetic resonance imaging (MRI), or histologic results were excluded. Reference lists from the selected papers were also screened. A flowchart of the systematic review is provided in Figure 1. Relevant data were then extracted and collected in a unique database, with the consensus of the two observers, to be analyzed for the purposes of the present manuscript.

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Figure 1 PRISMA flowchart resuming the papers’ selection process. PRP: Platelet-rich plasma.

The main findings of the present systematic review are that: (1) there is a paucity of clinical trials investigating the role of stem cells in promoting ACL healing, both in the treatment of partial and complete ACL tears. Therefore, no conclusive statement can be issued regarding the efficacy of this treatment approach; (2) despite the high number of RCTs, the role of PRP in ACL healing is still controversial, and it is not possible to fully endorse this biologic strategy to this particular indication; and (3) the use of biologic agents in partial ACL rupture is very limited, with just two clinical trials published, and therefore, the possibility of treating this kind of injuries by regenerative approaches remains an open question.

Biologic approaches to enhance healing of musculo-skeletal injuries are for sure a fashionable topic in the field of regenerative medicine. The overall brilliant results documented by in vitro and in vivo trials have stimulated their use in clinical practice, with different indications and targets, ranging from degenerative conditions (such as osteoarthritis and tendinopathies) to muscle and ligament injuries[8,9]. The first product that has encountered a large clinical application (together with a large commercial success) is PRP, whose use has been documented in papers published more than a decade ago. Despite being no more a “novel” treatment option, there is still uncertainty about its real effectiveness and, up to the present moment, there is no clear recommendation for its use in any of the clinical conditions. Therefore, we underline the fact once again, that the positive findings from pre-clinical studies cannot be reproduced in the in vivo model, whose complexity cannot be fully mirrored in laboratory experiments[40].

Looking at PRP potential in ACL healing, the literature is not conclusive with regards to the benefit of PRP application in providing a faster recovery and better functional outcome after ACL reconstruction. However, the evidence is currently limited to short term evaluations (up to 24 mo), whereas no trial has yet pointed out if the administration of platelet-derived growth factors could be effective at longer follow-up, providing more stable results or lower rate of recurrent injury. Interestingly, most of the studies documented a superior graft maturation over time after PRP administration. These findings were achieved by imaging evaluation, and in one case also by histology[39]. A better ligamentization of the graft may be related to superior and longer lasting mechanical properties that could impact clinical outcome at middle-to-long term evaluation. In light of that, further studies correlating imaging, histology and functional scores are expected to better clarify the role of PRP. Considering that patients after ACL reconstruction have high expectations in terms of durability of results, the possibility of reducing re-injury rate and providing a more stable clinical outcome seems very attractive and could justify the use of biologic agents. With regards to aspects such as bone-graft integration and prevention of bony tunnel enlargement, results are controversial and, overall, the majority of trials fail to support the use of PRP for this indication. By the way, research is moving forward in the attempt to optimize PRP technology to obtain the best possible results[41]. The main limiting factor that scientists and clinicians have to overcome is the great inter-product variability and the absence of a well defined therapeutic protocol[42]. When so many variables come into play, there is an intrinsic difficulty in finding the right way to move on. First of all, there are plenty of different PRP products that could be used. All of them differ in many fundamental aspects, such as preparation procedures, cellular content, platelet concentration rate, physical properties and eventual use of activators to enhance growth factors’ release. Authors have been using either lab made products or commercial kits from several companies, all characterized by different preparation protocols that rendered different PRP formulations[11,43,44]. In particular, the aspect of cellularity is currently the most debated one and there are controversial findings regarding the role of the different components of PRP, especially leukocytes whose presence has been deemed as detrimental based on laboratory findings that have not been confirmed in the clinical setting, leaving many questions open to investigation[45,46].

Another fundamental issue regards the application modalities of PRP during ACL reconstruction or repair. Several different approaches have been proposed, ranging from simple intra-articular injections to intra-ligamentary deposition, graft coating, topical use into the bony tunnels, or at the bone-graft interface. Even more complex strategies, such as suturing PRP clots or PRP-soaked sponges directly onto the graft have been suggested. In any case, based on the available data, it is impossible to endorse an ideal product or an ideal therapeutic modality to stimulate ACL healing.

Looking at the application of mesenchymal stem cells, they have been introduced into clinical practice more recently than PRP, and they have been tested mainly in the field of cartilage regeneration/osteoarthritis[47]. With regards to ACL healing, the current evidence is strongly affected by the paucity of literature available that prevents any indications for the use of such biological augmentation. As pointed out previously, only two papers in total (one RCT and one case series) investigated the potential efficacy of bone marrow concentrate in this particular field of sports medicine[17,18]. One of these studies applied the stem cell concentrate to treat partial ACL tears together with PRP, which prevents any clear understanding of the real contribution of each biological product. The lack of data is a severe flaw, also considering that stem cell therapy is characterized by the same great variability described in the case of PRP products. Many factors should be taken into account. First of all, the source of stem cells should be considered, since it is possible to obtain them from different anatomical sites (bone marrow, adipose tissue, synovial tissue, peripheral blood and so on), and this peculiar aspect could play a major role in determining clinical outcome. Furthermore, stem cells could be used as a concentrate, or could be expanded in vitro and then applied during surgery[48]. By the way, the application of cellular therapy in orthopaedics is under strict surveillance and clinicians have to face regulatory burdens that are currently limiting the number of ongoing clinical trials[49]. For this reason, there is such a great dichotomy between the flourishing pre-clinical literature and the very limited data coming from clinical studies. This explains the fact that mainly bone marrow concentrate has been tested for ACL healing at the moment, since it is the easiest and safest way to collect and administer stem cells in a surgical setting. In light of these remarks, both in the field of PRP and stem cell augmentation for ACL healing, there is a need of further basic science studies to better understand the mechanisms of action of these powerful biological agents. Moreover, there is also a need of more high-level comparative trials that could clarify if some specific “products” or applicative modalities are truly better than others.

A further consideration should be issued on the ACL injury patterns that have been treated with biologic agents. The large majority of papers (21) were focused on their application during ACL reconstructive surgery, whereas only two trials, both case series, have investigated their potential in partial ACL tears (Table 1). The possibility of enhancing ACL healing in case of partial rupture is very attractive, because it can contribute to functional recovery and avoid the higher surgical stress of traditional reconstruction. However, even in this case, the current lack of data prevents a reliable assessment of the efficacy of biologic agents, when applied for this specific purpose. The technique described in the literature to deliver PRP or stem cells into the injured ACL (intra-ligamentary injection under fluoroscopic or arthroscopic check) is feasible[18,19] and results seem to be encouraging. Again further high quality trials, with higher number of patients and longer follow-up, are needed to confirm these preliminary findings. Furthermore, considering that partial ACL tears may have variable features, it would also be clinically relevant to introduce and validate a classification system, with the aim of understanding whether the biological approach could be more effective in specific lesion patterns.

In conclusion, based on the available clinical evidence, there is a lack of data about the efficacy of stem cells in ACL healing, whereas the data concerning the role of PRP are not conclusive to understand if it could provide a faster recovery and better functional outcome during ACL repair/reconstruction. Despite some positive findings in terms of graft maturation and clinical outcome, further long-term studies are needed to identify whether the administration of PRP could truly play a beneficial role during ACL reconstruction. Lastly, in contrast to the large number of trials dealing with ACL reconstruction, the treatment of partial ACL tears with biologic agents has been poorly investigated, and therefore there is need of more high quality data to understand the efficacy of biologic agents in this particular application.