9.2.1 Preclinical Evidence

Preclinical basic science studies have elucidated many of the cellular and biochemical mechanisms with functional significance in biologic substances. These findings in turn have laid the groundwork for assessing the concentrations of important cytokines in biologic products and for evaluating the systemic effects of treatment. The α and dense granules in platelets store growth factors and other bioactive factors important for healing. When activated, platelets release growth factors contained in these α granules in a localized, site-specific manner. Growth factors that have demonstrated positive effects include bone morphogenetic protein, fibroblast growth factor, hepatocyte growth factor, insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), and vascular endothelial growth factor (VEGF). ⁸ However, other cytokines have demonstrated deleterious effects such as including interleukin 1 (IL-1), matrix metalloproteinases, tumor necrosis factor a. Dense granules release other bioactive molecules, such as serotonin, histamine, dopamine, calcium, and adenosine. Together, α and dense granules provide localized delivery of compounds that function in a complementary fashion during wound healing. ^{2 ,​ 3} Finally, MSCs provide numerous benefits including the ability to differentiate into one of multiple cell lineages, release growth factors, and mobilize the movement of stem cells during angiogenesis. ¹

To progress toward eventual human clinical studies, preclinical studies have been performed first to evaluate the safety and effectiveness of biological treatment strategies. For the most part, these models typically involve using in vitro basic science human or animal cell models or in vivo animal models. In addition, preclinical studies have facilitated the development and refinement of outcome measures that are later used in human clinical trials, including serum biomarkers and structural assessment tools such as imaging and histology. A general description of findings is presented in the following section.

9.2.2 Basic Science Evidence

Numerous basic science studies have been performed to evaluate the efficacy of biological treatments in promoting healing responses in the knee. These studies have used cellular models, either derived from humans or animals, to evaluate the cellular mechanisms that may have positive therapeutic effects following biologic treatment. De Mos et al ⁹ reported that PRP resulted in increased collagen and total cell proliferation, as well as matrix-degrading enzymes, in human tenocyte cultured cells, which they hypothesized may help the healing response of tendons. ⁹ In addition, Schnabel et al ¹⁰ cultured equine flexor digitorum superficialis tendon expiants in PRP and reported increased gene expression of collagen I, collagen III, and collagen oligomeric protein, without the increase of catabolic matrix-degrading proteins. ¹⁰ These authors also promoted the use of PRP to stimulate tendon healing. ¹⁰ With regard to cartilage repair, Fukumoto et al ¹¹ reported that TGF-βl and IGF-1, commonly found in PRP, synergistically promoted the chondrogenesis of MSCs, which was measured by quantifying the amount of cartilage in the explants, in an in vitro rabbit model. Finally, Ishida et al ¹² reported that rabbit meniscal cells exhibited upregulation of meniscal cell viability, including increased synthesis of DNA and sulfated glycosaminoglycans, in vitro after treatment with PRP.

9.2.3 Animal Model Evidence

Animal model studies have been used to demonstrate the proof of concept for new biologic treatment approaches. Given the promising results that have been described by in vitro basic science models, many studies have moved toward the evaluation of biologic knee treatments in animal models. Oftentimes, outcome metrics utilized in animal model studies mirror approach those later used in human clinical trials.

For patellar tendinopathy, Taylor et al ¹³ reported that an autologous blood product injection was safe in an in vivo rabbit model based on histological analysis, which demonstrated an angiogenic response without abnormal histological markers. Kajikawa et al ¹⁴ reported increased collagen 1 and collagen III and macrophage production on histological and immunohistological evaluation in rats after injecting PRP, indicative of the mobilization of tendon healing. In addition, Wilke et al ¹⁵ created 15 mm articular cartilage defects in the patellofemoral joint of horses and reported that injection of autogenous fibrin with MSCs increased the early cartilage healing response in comparison to a cartilage-only model after biopsy. Therefore, biopsy with histological analysis may likewise prove useful in human clinical trials following biologics treatment.

To evaluate the effect of biologics on meniscal healing, Ishida et al ¹² created a 1.5 mm defect in the avascular zone of meniscal tissue, and treated the meniscal tissue with PRP, enclosed in a gelatin hydrogel scaffold designed to gradually time release PRP. After 12 weeks in vivo, the PRP-treated group demonstrated more fibrochondrocytes on histology, which led the authors to propose that PRP may be able to stimulate healing of the avascular meniscal tears. ¹² However, Zellner et al ¹⁶ did not report improved tissue fill in rabbits with the application of MSCs and PRP, with a hyaluronancollagen scaffold, implanted into 2 mm defects in the avascular meniscal zone, at 6 or 12 weeks in comparison to a cell-free scaffold.

With regard to soft tissue usage, biologics have also been evaluated in preclinical animal models. Unlike human models, in which biomechanical or histological analysis of tissue is typically very difficult, animal models allow for the direct comparison of healing after in vivo biological treatment. One group has reported enhanced ACL primary repair in a porcine model with significantly improved yield and stiffness at 3 months after implantation of a collagen-scaffold complex, intended to provide structural support for a healing clot, in comparison to a suture only repair. ¹⁷ The same group also reported similar results in a canine model with increased biomechanical strength after use of a collagen-PRP scaffold to treat a surgically sectioned ACL in comparison to controls at 6 weeks. ¹⁸ In addition, two studies reported similar increased biomechanical strengths of the medial collateral ligament (MCL), and increased neovascularization after the addition of growth factors that are commonly found in PRP after 6 weeks of healing in rabbit models. ^{19 ,​ 20 ,​ 21}

Future animal models should also evaluate soft tissue healing at multiple time points using magnetic resonance imaging (MRI), such as those described by Biercevicz et al, ²² that can assess the signal intensity at numerous time points. ²² Increased and/or decreased signal intensity on MRI imaging can indicate the progression of healing in ligamentous healing and should be further investigated. As shown by Biercevicz et al, ²² decreased signal intensity is significantly correlated with increased structural properties of anterior cruciate ligaments (ACL) of grafts. Therefore, assessing outcomes following biologic treatment using MRI obtained at multiple time points can further enhance understanding of outcomes in animal models. ²² In summary, the outcome measures used in basic science and animal model studies represent excellent techniques to emulate in various settings across human clinical trials.

9.3.1 Clinical Outcome Scores

Subjective clinical outcomes consist of patient reported outcomes that are quantified using outcome scores. Examples of outcome scores include the Lysholm score to report patient function after knee surgery, ²³ the Tegner scale to document activity level, ²⁴ and the short form 36 (SF-36) health questionnaire. ²⁵ Some metrics, such as the International Knee Documentation Committee (IKDC) form, include both a subjective questionnaire and objective exam components. ²⁶ Other metrics are more specific. For example, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) ²⁷ and Knee injury and Osteoarthritis Outcome Score (KOOS) ²⁸ were developed to assess important patient-centric outcomes in osteoarthritis. A second example is the Victorian Institute of Sports Assessment (VISA) to document outcomes for patellar tendinopathy. ^{29 ,​ 30} Still other measures are symptom specific, for example, the visual analog scale (VAS) to document the intensity of a patient’s pain. ³¹ Finally, a patient satisfaction with outcome question is often used to report overall satisfaction following treatment. Together, these outcome scores represent important tools for assessing patient-reported outcomes following biologic treatment. In the future, these scores will need to be evaluated for reliability, validity, and responsiveness for specific conditions.

There are many examples in the literature on effective use of clinical outcome scores following biologic treatment in the knee. To begin, the effects of biologics on patellar tendinopathy have been examined in numerous clinical studies. Preliminary studies reported significantly improved clinical outcomes after a combination of dry needling and PRP or autologous blood injections using the SF-36, Tegner, VAS, and VISA outcome scores. ^{24 ,​ 32 ,​ 33} These studies involved numerous injections spaced at least 2 weeks apart; however, no control groups were compared. ^{24 ,​ 32 ,​ 33} Vetrano et al ³⁴ performed a randomized controlled trial comparing the effect of PRP injections of extracorporeal shock wave therapy. Both treatments significantly improved VAS and VISA clinical outcome scores at 2-, 6-, and 12-month follow-ups, with PRP resulting in significantly better outcomes than shock wave therapy at 6 and 12 months. ³⁴ In addition, Dragoo et al ²⁹ performed a randomized controlled trial comparing the effects of PRP and dry needling to dry needling alone. ²⁹ They reported significantly better results at 12 weeks with the PRP and dry needling in comparison to dry needling alone on the VISA outcomes, but these reported differences were not present at 26 weeks of follow-up. ²⁹

With regard to treating osteoarthritis, a level II prospective cohort study reported that intra-articular PRP injections resulted in improved outcomes on IKDC and VAS evaluations, in comparison to high- or low-molecular-weight hyaluronic acid at 6-month follow-up in a prospective comparative study. ³⁵ A recent randomized controlled trial described better WOMAC and VAS outcomes from single or double injections of PRP to treat osteoarthritis in comparison to a saline control. Results revealed adverse effects in 17% of patients receiving PRP treatments. ³⁶ In addition, a recent randomized controlled trial reported that patients who received injections of peripheral blood progenitor cells and hyaluronic acid did not result in significant differences in IKDC outcome scores in comparison to a hyaluronic acid control group. ³⁷

Finally, clinical outcome scores have been evaluated for soft tissue injuries. Three studies have evaluated clinical outcomes after ACL reconstruction. ^{38 ,​ 39 ,​ 40} All three studies reported no significant difference between the control groups and groups treated with PRP using variable clinical outcome scores at follow-up. At 6 months follow-up, Orrego et al ³⁸ reported no difference on IKDC or Lysholm clinical outcome scores, ³⁸ whereas Ventura et al ³⁹ reported no difference in the KOOS or Tegner scores. ³⁹ At 2-year follow-up, Nin et al ⁴⁰ reported no difference in VAS or IKDC scores between the control and PRP-treated groups. However, the lack of significant difference is not all that surprising due to the transtibial tunnel drilling for the control groups that would act as a source of platelets and MSCs functioning in effect as incidental biological treatments.

As shown earlier, there are currently numerous strategies for incorporating subjective clinical outcome measures in human clinical trials for the evaluation of biological treatments. The authors recommend using outcome metrics that are pathology-specific to achieve a more specific evaluation of outcome. However, at the same time, caution is necessary because these outcome metrics do not measure structural outcomes of the cartilage, tendon, or ligaments; therefore, supplementation of other metrics that specifically look at structural outcomes or other objective measures is advised.