Leukocyte and Platelet-Rich Plasma (L-PRP) in Tendon Models: A Systematic Review and Meta-Analysis of in vivo/in vitro Studies

Purpose To perform a systematic review on the application of leukocyte- and platelet-rich plasma (L-PRP) in tendon models by reviewing in vivo/in vitro studies. Methods The searches were performed via electronic databases including PubMed, Embase, and Cochrane Library up to September 2022 using the following keywords: ((tenocytes OR tendon OR tendinitis OR tendinosis OR tendinopathy OR tendon injury) AND (platelet-rich plasma OR PRP OR autologous conditioned plasma OR leukocyte- and platelet-rich plasma OR L-PRP OR leukocyte-richplatelet-rich plasma Lr-PRP)). Only in vitro and in vivo studies that assessed the potential effects of L-PRP on tendons and/or tenocytes are included in this study. Description of PRP, study design and methods, outcomes measured, and results are extracted from the data. Results A total of 17 studies (8 in vitro studies and 9 in vivo studies) are included. Thirteen studies (76%) reported leukocyte concentrations of L-PRP. Four studies (24%) reported the commercial kits. In in vitro studies, L-PRP demonstrated increased cell proliferation, cell migration, collagen synthesis, accelerated inflammation, and catabolic response in the short term. In addition, most in vivo studies indicated increased collagen type I content. According to in vivo studies reporting data, L-PRP reduced inflammation response in 71.0% of studies, while it enhanced the histological quality of tendons in 67.0% of studies. All 3 studies reporting data found increased biomechanical properties with L-PRP treatment. Conclusions Most evidence indicates that L-PRP has some potential effects on tendon healing compared to control. However, it appears that L-PRP works depending on the biological status of the damaged tendon. At an early stage, L-PRP may accelerate tendon healing, but at a later stage, it could be detrimental.


Introduction
Tendons, tight connective tissues, connect muscles and bones and transmit forces from the muscles to the bones, allowing the joint to move [1,2]. Terefore, the tendon bears heavy mechanical loads and is prone to injury, which can afect tendon function [3]. Tendon diseases are common clinical diseases, mostly in athletes and inactive people, which constitute about approximately 30-50% percent of all sports injuries [4].
Nowadays, platelet-rich plasma (PRP) has gained a lot of interest in the treatment of tendon injuries [5]. PRP is a platelet concentrate obtained from whole blood through centrifugation [6]. A large body of literature suggests that PRP may have multiple potentials for tendon repair and regeneration because they store and release extensive growth factors, such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), and basic fbroblast growth factor (bFGF), insulin-like growth factor (IGF) [7,8]. Tese growth factors are secreted by the dense granules, α-granules, and lysosomes in platelets [7,8]. In addition to growth factors, other components including plasma, leukocytes, and residual erythrocytes also contain and/or release quite a few bioactive factors [7].
Although several studies have reported favorable therapeutic outcomes with PRP, some studies have shown less favorable results. Tese conficting results are mainly ascribed to the diferent PRP preparation methods. A recent study investigated the cellular components of PRP and found signifcant diferences in leukocyte concentrations between PRP preparations contrasted to platelets and fbrinogen [9]. According to whether they had more or fewer leukocytes than autologous blood, PRP can be divided into leukocyte-and PRP (L-PRP) and pure PRP (P-PRP) [10][11][12][13][14]. Moreover, the concentration and components of leukocytes have a signifcant efect on the function of PRP [15]. Most of the past research has not identifed the specifc components of PRP; however, because of the controversy over the therapeutic efect of PRP, a growing number of studies have identifed the type of PRP based on whether it contains leukocytes or not [10,13,14,16].
A systematic review will be conducted to determine if the delivery of L-PRP has promoted tendon healing in terms of methodology and reporting of the outcome. Te mechanism of action of L-PRP and its efcacy in treating tendon injury will be studied in more detail and will ultimately demonstrate some benefts of L-PRP for tendon disorders in vitro and in vivo.

Literature Search.
We performed this literature review of in vivo/in vitro studies through foreign databases including PubMed, Embase, and Cochrane Library up to September 2022. Te search terms were a combination of medical subject heading (MeSH) terms and their synonyms. Te search query used was as follows: ((tenocytes OR tendon OR tendinosis OR tendinitis OR tendinopathy OR tendon injury) AND (platelet-rich plasma OR PRP OR autologous conditioned plasma OR leukocyte-and platelet-rich plasma OR L-PRP OR leukocyte-richplatelet-rich plasma OR Lr-PRP)).

Exclusion and Inclusion Criteria.
Only in vitro and in vivo studies that assessed the potential efects of L-PRP on tendons and/or tenocytes are included in our review. We carefully reviewed the specifc methodology of each included study, especially to accurately determine leukocyte concentration in the fnal PRP product used for tendon injection. L-PRP was characterized as PRP with a leukocyte concentration exceeding that of whole blood, whereas P-PRP was defned as PRP with a lower leukocyte concentration than that of whole blood [10][11][12][13][14]. When insufcient information was provided in the article, the study authors were contacted to acquire leukocyte concentrations. If the study authors did not record leukocyte concentrations, the manufacture's documentation of the PRP system they used was consulted to extract details about it. At the same time, according to the relevant literature analysis of PRP systems, it was decided which PRP system can produce L-PRP and included in this study, such as the Mini GPS III system, Smart PreP autologous platelet concentrate system, and platelet concentration collection system (PCCS), whereas the Arthrex autologous conditioned plasma doublesyringe system, the Selphyl system, and the Endoret systems are known to produce leukocyte-poor PRP [15,[17][18][19][20][21]. With these methods, all formulations can be clearly classifed into either class L or class P. Articles of undefned types of PRP and only P-PRP were excluded.
Of the studies that included additional therapeutic variables, only those trials that compared L-PRP directly to the control group (no treatment, saline solution, or control cell medium) were evaluated.
Te study excluded randomized controlled trials and case studies. Only English-language studies published in peer-reviewed journals were considered.
Two authors (YLL and MRY) searched separately, determined articles based on inclusion and exclusion criteria, and completed the PRISMA guidelines ( Figure 1). Studies that met the criteria were cross-checked and included. In case of discrepancies, discussions and decisions were made by the senior researcher.

Data Extraction.
Two authors extracted the article data and developed a standardized data table. Data collected included descriptions of PRP, study design and methods, outcomes measured, and results.
In vitro studies were analyzed for cell proliferation, cell migration, cell diferentiation, the content of collagen types I and III, infammatory mediation, and catabolic response. In vivo studies were analyzed for signal intensity in magnetic resonance imaging (MRI), collagen fbril diameters, histologic assessment of tendon repair, angiogenesis, infammatory mediation, the content of collagen types I and III, cross-sectional area (CSA), lesion percent of the involved tendon, and biomechanical testing.
Only 1 in vitro study reported the infuence on cell migration, which illustrated increased cell migration after the application of L-PRP [25].
Less is known about the infuence of L-PRP on cell diferentiation. Among the eight in vitro studies, 2 reported cell diferentiation data, with 1 study demonstrating signifcant increases in the diferentiation of TSCs into active tenocytes [29] and another demonstrating nontenocyte diferentiation of TSCs [28].
Te efect of L-PRP on collagen types I and III was reported in 7 studies, 6 of which showed signifcant increases in collagen I and III or collagen I/collagen III ratio [22-24, 26, 28, 29], and only 1 showed a signifcant decrease [25].
Signal intensity in MRI with L-PRP treatment is analyzed in 3 studies, 2 of the studies demonstrated a signifcant decrease in T2 mapping signal intensity in MRI [34,36], while the third study showed no change [38] (Table 4).
Tree studies reported data on collagen fbril diameters, and all of them demonstrated signifcant increases with the use of L-PRP [34,36,38].
Six of 9 in vivo studies reported histologic assessment of tendon repair after L-PRP treatment. Four of the studies reported that L-PRP signifcantly improved the quality of tendon injury tissue [34][35][36][37], whereas 2 studies demonstrated no change [32,38]. Tree studies reported data on angiogenesis, with 2 of the studies reporting L-PRP signifcantly accelerated angiogenesis of tendon, which then  Zhang et al. [28] Leukocytes in L-PRP were 4 times higher than whole blood; leukocytes in P-PRP were 2 times lower than whole blood TSCs L-PRP was prepared from rabbits. TSCs were isolated from the patellar tendons of healthy rabbits. TSCs cultured and treated with DMEM with FBS (control group), P-PRP, or  Evidence-Based Complementary and Alternative Medicine 5     Evidence-Based Complementary and Alternative Medicine 9  gradually decreases with the tendon healing process [35,37], whereas the third study showed a signifcant decrease [34]. Te efect of infammatory mediation with L-PRP treatment was most widely researched. Seven of the 9 in vivo studies reported infammatory mediation. Among them, 5 studies showed a signifcant decrease [31,33,34,36,37] (4 of them reported an initial increase, but a decrease over time), and 2 studies showed no diference [32,38].
Te efect of L-PRP on collagen types I and III was also analyzed in a small amount of literature. Tree studies reported collagen type I, 2 of which showed a signifcant increase [34,36] whereas the third study showed no difference [38]. Meanwhile, 2 studies reported collagen type III, and all of 2 showed a signifcant decrease [34,36].
Tree studies reported data on the cross-sectional area (CSA) or lesion percent of the involved tendon. Tree of them reported CSA, 2 of which showed a signifcant decrease [34,36], and 1 showed no diference [38]. Two studies reported lesion percent of the involved tendon, 1 showed a signifcant decrease [36], and 1 showed no diference [38].

Discussion
Nowadays, the application of PRP has progressed rapidly without a large amount of data to support its safety or clinical efcacy [39]. Although many meta-analyses have reported the clinical role of PRP, the results are still confusing, somewhat favorable, somewhat unhelpful, and somewhat even harmful [13,14,40,41]. Literature on PRP preparation methods as well as platelet concentration and cytology reports are inconsistent. Among them, leukocyte concentration is one of the most vital factors infuencing PRP function [13,15,24,40].
In this literature review, we searched basic science articles on the use of L-PRP on tendon disease. Unfortunately, because the number of studies included is too small, data could only be qualitatively analyzed. Further research must be conducted to support our fndings in the future.
In in vitro studies, parameters such as cell proliferation, the content of collagen types I and III, catabolic response and infammatory mediation have generally been used to assess the efcacy of L-PRP treatment for tendon repair, while in in vivo studies, the criteria of evaluation of L-PRP treatment are histologic assessment and infammatory mediation. Most evidence indicates that L-PRP has several benefcial efects on these parameters compared to control.
Four of 5 in vitro studies reporting cell proliferation showed a signifcant increase in the proliferative ability of tendon associated cells by L-PRP, and merely one showed a signifcant decrease. Tendons are rich in collagen, with the most abundant collagen being type I collagen, which accounts for approximately 95% of total collagen. Six of 7 in vitro studies reported collagen type I and III signifcant increase, and only one showed signifcant decrease. Meanwhile, in the in vivo studies, 2 of 3 studies that reported collagen type I demonstrated signifcant increases in the content of collagen type I, with the remaining 1 study showing no diference. Both 2 in vivo studies reported that L-PRP signifcantly decreases the content of collagen types III. After tendon injury, collagen type I content is downregulated and the synthesis of type III collagen is enhanced. As the tendon heals, type III collagen gradually converts into collagen type I. Tus, it seems that L-PRP could promote collagen type I synthesis but not collagen type III to facilitate tendon repair in both in vivo and in vitro studies.
Furthermore, all of 3 in vivo studies that reported collagen fbril diameters demonstrated that L-PRP signifcantly increases collagen fbril diameters. In in vivo studies, 4 of the 6 studies that reported the histological changes of tendons showed a signifcant increase in the quality of tendon healing by L-PRP, and the other 2 studies reported no diference. Parameters used to assess the quality of tendon histological repair include better fber structure and arrangement, less cell density, less angiogenesis, and less infammation.
Four of the 5 in vitro studies reported that L-PRP signifcantly increases catabolic cytokines, such as matrix metalloproteinases-9/11 (MMP-1/9). MMPs are zinc endopeptidases that regulate the extracellular matrix components, which may inhibit matrix formation. Some literature have proposed that L-PRP could have both catabolic and anabolic properties [31,42]. Nevertheless, this function appears to be time-dependent as there is less beneft in delaying L-PRP administration after the early injury.
All the 5 in vitro studies reporting infammatory mediation revealed that L-PRP signifcantly exacerbates infammation in a short time. However, 5 of 7 in vivo studies demonstrated that L-PRP signifcantly decreases infammation, 4 of which showed a short term increase but a long-term decrease while the other 2 studies showed no diference. Consistent with infammation mediation, 2 of  3 in vivo studies reported that L-PRP signifcantly decrease signal intensity in MRI in T2 mapping, which means a decrease in local infammation. It is worth noting that in these two works of literature, animal tendon injury models were established by injection of collagenase, and L-PRP treatment was applied 1 week after collagenase injection, which is generally considered to be the acute stage of tendon injury or the early stage of tendinopathy. However, when L-PRP treatment was applied 4 weeks after collagenase injection (usually considered to be later stageof tendon injury), there were no signifcant infammation changes observed by both MRI T2 mapping and infammatory mediation in histology. Interestingly, this is consistent with the histologic assessment of tendon repair. Te application of L-PRP to the early phase of collagenase injection improved the histology results, while there was no diference when applied to the later phase of collagenase injection. It demonstrates that the efects of L-PRP are multifactorial, and the timing of application may alter its anti-infammatory capacity and overall efcacy. Infammation is an essential process for tendon healing, and leukocytes contained in L-PRP are instrumental. During the infammatory phase, infammatory cells such as neutrophils, monocytes, and macrophages migrate to the damaged tissue and remove necrotic material by phagocytosis [43][44][45]. Several studies have shown that early administration of L-PRP accelerates the repair of tendinopathy in rabbits more than late administration, suggesting that L-PRP may promote infammation in the early stage to accelerate tendon healing, while the benefcial efect of L-PRP in the late stage of tendon lesion was not obvious [29,34,36,38].
Finally, for tendon function, all 3 in vivo studies demonstrated improved biomechanical testing, such as the failure load, stifness, and ultimate tendon stress, meaning that L-PRP improves tendon outcomes and allows the tendon to return to life and exercise earlier.

Limitations
In this study, the lack of uniform methodology and reporting of results was the major limitation for more detailed and indepth analysis and comparison, as previously described. Tis study also lacked a risk of bias assessment for the included studies. However, there is currently no efective tool to assess the existence of bias in basic scientifc research. A tool that could provide a more objective assessment of our topic should be available to evaluate the quality and bias risks of these basic scientifc studies.
In vivo and in vitro studies also have some inherent limitations. For in vivo studies, tendon injuries in animals are distinctly diferent from those in humans. In animals, the lesions are usually small, and the tendon thickness is thinner. In addition, animal models with surgical incisions or injections of collagenase cannot truly mimic clinical human tendon disease.
Because of these limitations, it is hard to apply our fndings to the clinic. However, basic scientifc research remains essential to evaluate the efcacy and mechanisms of L-PRP for tendon therapy.

Conclusions
In this review of the literature, it was found that L-PRP is benefcial to these parameters in comparison with controls, including angiogenesis, collagen synthesis, infammation, and biomechanical property. It appears that L-PRP works depending on the biological status of the damaged tendon. At an early stage, L-PRP may accelerate tendon healing, but at a later stage, it could be detrimental. Furthermore, study methodology, including the timing of PRP administration, is not standardized across studies, which hinders comparisons of the efcacy of L-PRP.
For a better understanding of L-PRP's role in tendon pathology, more rigorous controlled experiments and consistent evaluation criteria must be set up in future basic scientifc and clinical studies.

Data Availability
All data generated or analyzed during this study are included in this article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.