8.1 Introduction

Intra-articular tissues such as the anterior cruciate ligament (ACL) and meniscus have an intrinsically poor healing capacity. This has led to an intense interest in discovering methods to augment the biological responsiveness of cells in these tissues. Platelet-rich plasma (PRP) contains various growth factors, including transforming growth factor β-1 (TGF-β l), fibroblast growth factor-2 (FGF-2), insulin-like growth factor (IGF-1), epidermal growth factor, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) that have demonstrated positive effects on cell proliferation, cell migration, angiogenesis, and extracellular matrix production in numerous cell types both in vivo and in vitro models. ^{1 ,​ 2 ,​ 3 ,​ 4 ,​ 5} The primary cell in the ACL is the fibroblast. The fibroblast has receptors for many of the growth factors released by platelets, including PDGF, TGF-β , and FGF. PDGF (Table 8.1) stimulates fibroblast growth, migration, and biosynthetic activity. ⁶ Similar effects are seen with TGF-β and FGF. In vitro, ACL cell migration has been stimulated by TGF-β l, while PDGF and FGF stimulate cell proliferation in a three-dimensional (3D) collagen scaffold. ⁷ In this review, we will explore the current data relating to the use of PRP and related materials in the augmentation of ACL and meniscus healing.

The current surgical treatment for ACL injuries is ACL reconstruction where the torn ACL is replaced with a graft of the tendon. However, recent research has focused on the use of platelets and PRP to heal the injured ligament, rather than replace it. These studies have not yet progressed to a clinical trial, and in this section we will review the basic science behind the preclinical development of this new technique.

Previous studies have demonstrated that ligaments which exist outside of joints (extra-articular) heal with an orderly progression of events. The first basic process is bleeding and then formation of a fibrin-platelet clot within the wound site, which fills in the gap between the torn ends of the tissue and forms a provisional scaffold for the surrounding cells to move into and remodel into a functional scar. However, in the intra-articular environment, after an injury, there is an upregulated production of urokinase plasminogen activator by synoviocytes (Fig. 8.1), which converts the inactive plasminogen molecule present in synovial fluid into its active form, plasmin. ⁸ Plasmin quickly degrades fibrin. Therefore, if a tissue is exposed to synovial fluid after injury, the ends may bleed, but the fibrin is unable to form a stable clot as it is degraded too quickly. ^{9 ,​ 10 ,​ 11} The early loss of this provisional scaffold has been thought to be a major reason why tissues within joints, such as the ACL or meniscus, fail to heal after the injury. ^{9 ,​ 10}

PRP has been evaluated in animal models to stimulate healing of the ACL with suture repair; unfortunately, it has been found to be ineffective. ¹² In a larger animal study using 4-month-old Yorkshire pigs, the addition of a 3 x PRP to the suture repairs did not improve knee laxity or the maximum tensile load of the suture repairs after 14 weeks in vivo. The strength of all repairs at 14 weeks was less than 11% of the intact ligaments, suggesting minimal healing with all repairs. ¹² The use of PRP therefore, may have failed because the main structural protein within PRP is also fibrin. Therefore, the fibrin in the PRP is degraded by the active plasmin within the joint and the PRP is unable to stay in the ACL wound site. Interestingly, when collagen is combined with fibrin, a copolymer is formed which is resistant to degradation by plasmin. ¹³ Further studies using a collagen-based scaffold material to deliver the PRP have had greater success in stimulating ACL healing in animal models as the carrier stabilizes and protects the fibrin and platelets in the ACL wound site. ^{14 ,​ 15 ,​ 16}

8.2 Collagen—Platelet Composites in Anterior Cruciate Ligament Repair

While the use of PRP alone was ineffective at stimulating ACL healing and repair, the use of PRP in combination with an extracellular matrix protein scaffold containing collagen has resulted in a new technique for ACL treatment. This treatment of “bio-enhanced ACL repair” has been evaluated in two recent large animal studies in the porcine model, both of which reported no significant difference in mechanical properties when the bio-enhanced ACL repair and ACL reconstruction using an allograft tendon were compared at 3 months ¹⁵ and 1 year ¹⁵ after the surgery. The mean values for yield load were identical in the repaired and reconstructed groups at 3 months after the surgery, while the mean values for maximum load and linear stiffness were higher in the bioenhanced repair group at 3 months; however, these differences were not statistically significant, possibly due in part to the variability seen in both the groups. ¹⁵ In addition, at 3 months, the mean values for the anteroposterior laxity of the knee were lower in the bio-enhanced ACL repair group than in the ACL reconstructed group, although again, these differences were not statistically significant, which may also be due in part to the variability in both the groups. ¹⁵ For the porcine knees studied at 1 year after ACL repair (Fig. 8.1) using bioenhancement with an extracellular matrix scaffold loaded with whole blood resulted in repairs with the same maximum load, stiffness, and knee laxity as animals treated with a bone-patellar tendon-bone allograft, but the animals treated with bio-enhanced repair had no more cartilage damage at 1 year than the contralateral knee, while the animals treated with ACL reconstruction had large lesions noted, particularly on the medial femoral condyle, ¹⁵ which is the same anatomic location seen in human patients at 10 to 15 years after ACL reconstruction.

The 1-year study in the porcine model referred to above ¹⁵ used whole blood as the biological adjuvant. It is possible that the results would be even better if a higher concentration of platelets were used in the form of PRP placed within a carrier to allow it to persist for a long enough period in the wound site. It has previously been reported that biological relationships are not always linear, but are often confined to a small window of effectiveness (i.e., the dose-response curve is not linear). In addition, as PRP is a stimulator of other cells, a higher concentration may or may not be helpful because its effects are dependent on the presence of responsive cells. ¹⁷ If only a limited number of cells capable of responding are available, then the highest concentration of PRP may not be able to produce a result better than a lower concentration. Finally, while there is evidence for many positive effects of PRP on wound healing, there is also an evidence for some negative effects. ^{18 ,​ 19 ,​ 20 ,​ 21} It is not known how higher concentrations of platelets affect the balance between positive and negative effects of PRP. ^{18 ,​ 20 ,​ 21}

Experimental data examining the effect of various concentrations of platelets in PRP on ACL cells in vitro and in vivo have been performed. The use of higher concentrations of platelets (3x or 5x) in vitro were not as effective as lx platelet preparations at simulating types I and III collagen gene expression and cell metabolism and preventing apoptosis. ²² Increasing the platelet concentration in vivo from 3x to 5x did not result in any improvement in the structural properties of the healing ACL, ²³ although the group treated with 5x PRP did have a higher cell number seen in the healing tissue. While not statistically significant, the means of the mechanical properties of the group treated with the 3x platelet concentration were better than the 5x group. Thus, in vitro and in vivo data currently suggests that the use of concentrations of platelets greater than 1x within extracellular matrix scaffolds may not be as effective at stimulating ACL healing as physiologic concentrations. However, the inclusion of plasma with platelets has been found to improve ACL fibroblast collagen gene expression. In vitro studies looking at the combination of platelets and plasma on ACL cells have found that while exposing ACL fibroblasts to platelets or plasma resulted in decreased cell death and an increased metabolic activity of the cells in a collagen hydrogel, the combination of both platelets and plasma was required to stimulate type I and type III collagen gene expression. ²⁴

In summary, suture repair of the ACL is not improved with the use of PRP alone, but the ACL can be effectively repaired with the use of whole blood containing a physiological concentration of platelets in an extracellular matrix-based scaffold. The results of this bio-enhanced repair technique are similar to ACL reconstruction in terms of the mechanical properties of the healing tissue and graft, but the bio-enhanced repairs resulted in less post-traumatic osteoarthritis in large animals.

8.3 Anterior Cruciate Ligament Reconstruction and Platelet-Rich Plasma

The use of platelets to improve the mechanical properties and performance of an ACL graft seems a reasonable approach to improving outcomes of this operation. As noted above, when platelets are combined with collagen, the combination can help heal partial and complete transections of the ACL. ^{25 ,​ 26 ,​ 27 ,​ 28} Thus, it seems reasonable to assume that a platelet-based preparation could also stimulate ACL graft healing.

Use of a collagen-platelet composite to enhance ACL graft healing has recently been studied in large animal models. In goats, the use of a collagen-platelet composite resulted in a 30% reduction in knee laxity over grafts treated with a collagen scaffold alone. ²⁹ The platelet preparation used in this study was whole blood (1x platelets).

Another study in pigs evaluated the performance of a 5x PRP solution in combination with a collagen scaffold on graft healing in immature animals. ³⁰ Significant improvements in the graft tensile properties were found with the use of the 5x PRP-collagen composite with a 60% increase in failure load of the graft and decreased laxity of the PRP knees at 3 months after surgery. ³⁰ These data provide encouragement regarding the efficacy of the platelet-enhanced ACL reconstruction approach in immature animals.

However, when a similar study was performed in adolescent animals, the results were not as promising. The use of a 1 x PRP-collagen composite resulted in improved stiffness of the graft, but higher concentrations (3x and 5x) did not. The use of the collagen-platelet composite did, however, result in less cartilage damage to the knees when evaluated at 3 months after surgery than the knees treated with graft reconstruction alone. ³¹

While no clinical trials for the use of collagen-platelet composites have been conducted to date, several clinical studies evaluating the effect of the addition of exogenous platelets on graft healing have been performed. A recent systematic review identified eight studies, seven of which focused on graft maturation and five on bone-tendon healing in the tunnel. ³² Four of the seven studies reported significantly better maturation “outcomes” in the grafts treated with concentrated platelets compared with those that were not. ^{33 ,​ 34 ,​ 35 ,​ 36} In one study, evaluation of the grafts with magnetic resonance imaging (MRI) revealed that the signal intensity of a graft treated with 9x platelet solution at the time of surgery matched that of the intact PCL in 100% of the patients as compared with only 78% of those treated with a graft alone at 6 months after surgery. ³³ However, no differences in clinical outcomes between the treated and untreated groups were reported. ³³ A second study using MRI also found that the use of a 9x platelet concentrate reduced the average time to achieve a normal MRI signal intensity value from 369 to 177 days. ³⁵ Qualitatively, it has been observed that grafts treated with concentrated platelets (3x) showed higher arthroscopic ratings for synovial coverage, graft width, and graft tension in the platelet-treated group when compared with the controls. ³⁴ Histology revealed that the ligament maturity index ²⁶ was significantly greater in the platelet-treated grafts. ³⁴ Other studies have found no differences between the grafts treated with platelets versus untreated grafts in any outcome measures. ^{37 ,​ 38 ,​ 39}

The majority of studies evaluating graft-bone healing have found no improvements in healing at the graft-bone junction. ^{33 ,​ 36 ,​ 38 ,​ 40} In summary, the review of the literature suggests that the use of platelet concentrates may improve the rate at which grafts achieve low-signal intensity on MRI or an improved ligament maturity index on histology ^{33 ,​ 34 ,​ 35} ; however, none of them showed an improvement of clinical or patient oriented outcome even at 2 years, ^{32 ,​ 33 ,​ 36 ,​ 39} or significantly improved bone-tendon healing. ^{33 ,​ 36 ,​ 38 ,​ 40}

The results of the preliminary studies provide credence to the concept of bio-enhancing healing of an ACL graft with the application of platelets at the time of surgery. However, only two of the eight clinical studies that have been published meet the standards of level 1 evidence and none of them report long-term outcomes (> 2 years). Thus, the effects of these concentrated platelet preparations on cartilage health and overall knee joint function after ACL injury and reconstruction remain unknown. In addition, the PRP and platelet concentrate formulations used in the various studies differed not only in platelet concentration, but also in the concentration of white blood cells and PRP activation technique. It is extremely likely that the other blood constituents in some of the preparations of PRP (i.e., leukocytes, erythrocytes) may be important in ACL graft healing. ⁴¹ As noted earlier, for ACL repair, the delivery of the platelet preparation may also prove to be important for graft healing as well. Future studies will be required to find the best way to deliver a platelet preparation, as well as how to optimize the biologic adjunct to maximally stimulate the healing response of the graft, and to improve the long-term outcomes after ACL reconstruction, particularly in terms of preventing the development of post-traumatic osteoarthritis.

8.4 Biological Enhancement of Meniscal Restoration

The meniscus functions in the knee joint by distributing load, improving congruency, enhancing stability, and provides joint lubrication contributing to the reproducible articulation between the femur and the tibia. ^{2 ,​ 5} When the integrity of the meniscus is disrupted, contact stresses are increased, leading to a well-established association with osteoarthritis. ^{42 ,​ 43 ,​ 44 ,​ 45} Thus, current treatment paradigms advocate for meniscal restoration where possible and biological strategies have focused on expanding operative intervention to allow repair and healing of tears with unfavorable healing characteristics. ⁵ The use of exogenous fibrin clot is representative of earlier augmentation techniques in meniscal repair and has demonstrated some success in animal and clinical studies despite the technical difficulties of keeping the clot in situ at the repair site. ^{46 ,​ 47 ,​ 48 ,​ 49}

Vascularization of the meniscus is inextricably linked to the success of meniscal repair and delineates zonal variations in cellular and biochemical composition of the meniscal tissue. ² During prenatal development the meniscus is a fully vascularized and relatively homogenous tissue; during childhood, there is a regression of the peripheral vascularization. By the age of 10 years, the peripheral (10–30%) meniscus is vascularized and this changes minimally to 10 to 25% by skeletal maturity. ⁵⁰ For the purpose of preclinical study and clinical correlation, the meniscus has been divided into three zones: the red–red zone (vascularized), red–white zone (partial vascularization), and white–white zone (avascular). Biological augmentation techniques have been focused on improving clinical outcome for tears that extend into the avascular zone. ⁵¹

When specific tear or patient characteristics are unfavorable for primary repair, partial or total menisectomy is often undertaken to relieve patient symptoms. ⁵² In orthopedic centers currently outside the United States, biodegradable meniscal scaffolds (Collagen meniscus implant, Ivy Sports Medicine, Montvale, NJ; Actifit, Orteq Limited, London, United Kingdom) are implanted immediately following partial menisectomy in knees that demonstrate minimal degenerative cartilage changes; ligamentous repair, cartilage restoration, and realignment procedures may be performed concomitantly. ⁵ The goal of implanting a meniscal scaffold is to promote meniscal tissue regeneration on a 3D matrix in the defect space. Scaffolds are fixed to adjacent meniscal tissue from the peripheral rim through the body of the meniscus using sutures; the meniscal-scaffold interface often extends from the red-red vascularized zone to the inner white–white avascular zone. Cellular infiltration and matrix deposition across the meniscus–scaffold interface (constituting biological integration) are critical for the success of meniscal scaffolds that have been associated with promising clinical results. ^{53 ,​ 54 ,​ 55 ,​ 56} Biological enhancement of meniscal repair at the meniscus-meniscus tissue interface shares common biological goals with meniscal regeneration at the meniscus-scaffold interface in terms of cellular migration and matrix deposition at the regeneration site and warrants consideration in anticipation of biological augmentation of meniscal scaffold technology.

The primary cell in the meniscus is the fibrochondrocyte. However, fibrochondrocytes are subject to zonal variations in morphology and behavior likely relating to the surrounding matrix and the presence of vascularity. ⁵⁷ Inner white-white zone fibrochondrocytes are more chondrocyte-like and are situated in a glycosaminoglycan (GAG) rich matrix that has higher levels of type II collagen (relative to the rest of the meniscus) and approximate the composition of articular cartilage. In contrast, the outer meniscus contains fibrochondrocytes that are more fibroblast-like in a vascularized matrix consisting of mainly type I collagen and relatively less GAG than the inner zones. ² In addition, a surface layer fibrochondrocyte cell type has also been described. ⁵⁷ Accordingly, many in vitro studies have differentially tested fibrochondrocytes by zone when assessing cell behavior and this is important in the translational interpretation of preclinical in vitro testing of biologics toward improved meniscal repair. ^{4 ,​ 57 ,​ 58 ,​ 59}