Abstract
Background:
Platelet-rich plasma is reported to contain multiple growth factors, and has been utilized in orthopaedic surgery to aid healing in multiple tissues. To date, the use of autologous platelet-rich plasma has not been studied for its effects on normal soft tissue.
Methods:
Eighteen adult New Zealand White rabbits were injected with 0.5 mL of autologous platelet-rich plasma in the right or left quadriceps muscle, Achilles tendon, medial collateral ligament, subcutaneous tissue, tibial periosteum, and ankle joint. Saline solution was injected on the contralateral side as a control. The soft tissues were examined histologically at two weeks (six rabbits) and six weeks (six rabbits), and soft tissues from six rabbits that had been reinjected at six weeks were examined at twelve weeks.
Results:
Inflammatory skin lesions were visible at forty-eight hours at superficial platelet-rich plasma sites. All lesions resolved by six days. Compared with findings in control specimens, histological analysis of platelet-rich plasma injection sites at two weeks showed a marked inflammatory infiltrate with lymphocytic and monocytic predominance. Intra-articular injection showed villous synovial hyperplasia and chronic synovitis. Tendon and ligament sites showed new collagen deposition. Intramuscular injection sites showed thrombosis, necrosis, and calcium deposition. Subcutaneous sites also showed calcium deposition without necrosis as well as collagen nodules representing early scar tissue. Histological examination of platelet-rich plasma injection sites at six and twelve weeks demonstrated a persistent but diminished inflammatory infiltrate. Focal areas of scar tissue were seen with fibroblasts, collagen formation, and neovascularity. All saline solution sites at all times were nonreactive.
Conclusions:
Platelet-rich plasma can initiate an inflammatory response in the absence of an inciting injury in normal soft tissue in rabbits.
Clinical Relevance:
Platelet-rich plasma has gained widespread use clinically in the treatment of a variety of orthopaedic injuries and as a surgical adjunct; however, its in vivo effect on normal tissues has not been examined in a controlled laboratory study.
The use of biologics in orthopaedic surgery has undergone a rapid evolution. Growth factors have been isolated and are gaining use in the promotion of soft-tissue and bone healing. Autologous platelet-rich plasma is an appealing source of growth factors. These growth factors include platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β), insulin-like growth factor (IGF), epidermal growth factor (EGF), fibroblast growth factor (FGF-2), and connective tissue growth factor (CTGF)1-4. Platelets also contain other cytokines and chemical mediators including histamine, fibrinogen, fibronectin, serotonin, complement c5a, and von Willebrand factor. Unlike newly released drugs that must undergo clinical trials and Food and Drug Administration approval, platelet-rich plasma is an autologous blood-derived product. Its use was initially described in the dental literature as a topically applied bone-graft supplement5. Its early use in orthopaedic surgery was expanded from a bone-graft adjunct to soft-tissue applications without rigorous data to support its safety and efficacy.
The purpose of this study was to examine the effects of platelet-rich plasma injection into various soft tissues. Our hypothesis was that platelet-rich plasma would initiate a different histological response in soft tissue compared with saline solution injections.
This study was undertaken after approval from the University of Colorado Animal Care and Use Committee. Animals were cared for under the direction of the staff veterinarian for the University of Colorado Animal Care Facility.
Eighteen skeletally mature male New Zealand White rabbits were anesthetized with use of ketamine (25 mg/kg) and xylazine (5 mg/kg). Autologous platelet gel was prepared in the following manner: (1) 50 mL of blood was phlebotomized via an ear vein from each rabbit with 6 mL of anticoagulant (ACD-A; anticoagulant citrate dextrose solution A); (2) whole citrated blood was loaded into a plastic disposable device consisting of two flexible bags affixed to a rigid plastic top and connected by a single plastic tube to affect fluid transfer (Platelet Concentrate Collection System [PCCS] tube; 3i Implant Innovations, a Biomet Company, Palm Beach Gardens, Florida) and spun at 3000 rpm for three minutes and forty-five seconds to separate plasma and platelets from red cells, (3) plasma and platelets were transferred to the other chamber of the PCCS disposable device and were spun at 3000 rpm for thirteen minutes to pellet platelets and white blood cells; and (4) platelet-poor plasma was removed and the platelet plug was resuspended in plasma to a total volume of 2.5 mL. To activate the resulting platelet-rich plasma, 5000 U of topical thrombin (bovine origin, Thrombin-JMI; GenTrac, Middleton, Wisconsin) was mixed with 30 mL of CaCl, and 0.5 mL of that mixture was added to 2.5 mL of platelet-rich plasma, yielding 3.0 mL of autologous platelet gel. Autologous platelet gel was injected in 0.5-mL volumes into six prepared sites. Injection sites included the quadriceps muscle, inguinal subcutaneous tissue, the Achilles tendon, the medial collateral ligament (MCL), the tibial periosteum, and the hock (ankle) joint. Each animal served as its own control, with one side injected with a 0.5-mL volume of platelet-rich plasma and the other with 0.5 mL of normal saline solution into each test site. The right and left legs were randomly assigned, and injection locations were marked with skin tattoos for accurate histological sampling. The eighteen animals were divided into three groups of six animals: group 1 was killed at two weeks; group 2, at six weeks; and group 3 was reinjected at six weeks and killed at twelve weeks. A reinjection group (group 3) was chosen to mimic a potential clinical situation to determine whether a so-called booster effect may be obtained by repeated treatment with platelet-rich plasma.
Finally, after calcification was observed within muscular and subcutaneous tissues in the initial three groups, a fourth group of six rabbits was injected with use of a calcium-free solution in order to assess what effect exogenous calcium from the activator may play in the development of soft-tissue calcific deposition. Platelet-rich plasma was prepared in a similar manner; however, the activator was created by mixing 5000 U of topical thrombin with 30 mL of 0.9% sodium chloride. We added 0.2 mL of the NaCl-thrombin activator to 0.8 mL of platelet-rich plasma, and 0.5 mL of this mixture was injected into muscle and subcutaneous tissue each. A randomly assigned control side was injected with 0.5 mL of platelet-rich plasma without activator to assess any adverse effect of the thrombin. These animals were killed at two weeks for histological evaluation.
The animals were killed with an anesthetic overdose (25 mg/kg of ketamine, 5 mg/kg of xylazine, and 260 mg of phenobarbital). Histological examination was carried out in a blinded manner by a board-certified musculoskeletal pathologist. Tissues were embedded in paraffin and stained with hematoxylin and eosin, trichrome, and azure stains to assess inflammatory cell infiltrate, neovascularization, and new collagen formation. Von Kossa and alizarin red-S stains were used to identify calcium deposition within soft tissues.
Source of Funding
Funding for this study was provided by a grant from the William H. Hurt Family Foundation and the Aspen Sports Medicine Foundation.
Platelet-Rich Plasma
Serum platelet count was measured in six of the eighteen animals (see Appendix). Average serum platelet count (and standard deviation) was 189,333 ± 47,047. Platelet count in the platelet-rich plasma for these animals averaged 1,348,667 ± 427,278, yielding a 712% concentration of the average serum platelet count.
Gross Examination
Seventeen of eighteen animals exhibited raised red skin lesions at superficial platelet-rich plasma injection sites (subcutaneous tissue, MCL, and periosteum) starting at postinjection day 2, enlarging by day 6, and gradually resolving. No treatment was administered for any lesion. Deeper muscular and intra-articular injections showed no superficial reaction. All saline solution sites remained normal on visual inspection. No changes in animal behavior were noted in either group.
Histological Analysis
Muscle
Two-week specimens were noted to have a monocytic and lymphocytic infiltrate with edema and necrosis noted in the muscle fibers. Deposition of calcium was present within the muscle and collagen fibers. At six weeks, the inflammatory infiltrate persisted with the addition of multinuclear giant cells. Fibrosis, muscle necrosis, and calcium deposition was seen (Fig. 1). In the area believed to represent the site of platelet-rich plasma injection, calcification of individual damaged muscle fibers was seen, and at later stages, these fibers were being actively reabsorbed by surrounding multinucleated giant cells. At twelve weeks, no significant increase in inflammation was evident. All saline solution muscle injection sites had normal findings on histological examination.
Animals injected with calcium-free activator also demonstrated large areas of muscle necrosis and vascular thrombosis. Calcification was noted in only one of six specimens, appearing in closer proximity to the periphery of the injection site (Fig. 2).
Subcutaneous Tissue
Platelet-rich plasma injection sites at two weeks demonstrated collagen nodules within the subcutaneous tissue, as well as new fibrous tissue including collagen fibers, and fibroblasts (Fig. 3). Subcutaneous fat was replaced with fibrous and monocytic inflammatory infiltrate (Fig. 4). At six weeks, chronic inflammatory cells persisted adjacent to calcium deposits within the dermis. Microcalcification was present in four of the six animals at two weeks and in one animal at six weeks; it was not seen at twelve weeks. Disorganized new collagen fibers were present. At twelve weeks, the inflammation was less predominant and inconsistent between animals. Animals injected with calcium-free activator showed no evidence of calcium deposition.
All saline solution injection sites in subcutaneous tissue had histologically normal findings.
Tendon
Platelet-rich plasma injected within the Achilles tendon showed monocytic and lymphocytic inflammatory cells with marked thickening of the peritenon at two weeks (Fig. 5). Tendon structure showed areas of basophilic infiltrate and vacuole formation with associated multinucleated giant cells. The von Kossa stain demonstrated no calcification within tendon tissue. A trichrome stain verified the presence of new collagen bundles (Fig. 6). At six weeks, chronic inflammation continued to be noted in the peritenon without obvious changes within the tendon structure itself. At twelve weeks, this response was decreased despite reinjection. In twelve-week specimens, three of six animals had calcified lesions similar to those in muscles, in what appeared to be sites where tendon attached to muscle. This was unique to this group and may be associated with the fact that they were reinjected. All saline solution injection sites in tendon were histologically normal.
Joint
Histological examination of the intra-articular injection site at two weeks showed villous synovial hyperplasia and marked chronic synovitis. No calcification was noted. However, because of the adjacent osseous structure, these specimens had been decalcified during preparation. In four of six animals, there were multinucleated giant-cell and histiocyte responses histologically similar to the subcutaneous calcification sites. At six weeks, one joint treated with platelet-rich plasma had a scarred nodule with multinucleated giant cells, and another had a fibrous nodule without multinucleated giant cells. The remaining six and twelve-week specimens had normal findings except for a single benign lymphoid nodule in a twelve-week specimen. At all time frames, the appearance of the articular cartilage was histologically normal with viable chondrocytes and normal matrix staining (Fig. 7). All sites of saline solution injection in joints were histologically normal.
Ligament
At two weeks, the MCL showed a monocytic and lymphocytic inflammatory infiltrate and thickening of the soft tissues. At six and twelve weeks, inflammation persisted but was much less prominent (see Appendix). All sites of saline solution injection in ligament were histologically normal.
Periosteum
At all time frames, there was no evidence of periosteal new bone formation with platelet-rich plasma injection. There was inconsistent hypercellularity of the periosteal tissue at two, six, and twelve weeks (see Appendix). Adjacent muscle tissue showed changes similar to those noted in quadriceps injection sites. All sites of saline solution injection in the periosteum were histologically normal.
Platelet-rich plasma has been used for multiple clinical applications in orthopaedic surgery, including wound hemostasis, wound sealing6,7, augmentation of bone grafts5,8-16, healing of chronic and acute wounds1,17-22, and treatment of tendinosis23-25. Use of platelet-rich plasma in total joint arthroplasty has shown decreased blood loss, decreased pain, and improved range of motion7. Platelet-rich plasma has been used in the treatment of delayed union and nonunions8, both alone and in combination with traditional bone grafts. The addition of platelet-rich plasma to a bone graft can increase bone density up to 20%12. Increased epithelialization has been demonstrated in both acute traumatic wounds18,19 as well as chronic diabetic wounds with use of platelet-rich plasma1,17,20-22. Platelet-rich plasma has been used clinically in the treatment of chronic tendinosis23-25. In addition, platelet-rich plasma has promoted tendon-healing in acute tendon injury and repair models2-4,26,27.
These diverse biologic effects associated with platelet-rich plasma may be explained at least in part by the variety of growth factors present in the α granules of platelets, and the fact that most of these factors may have actions on multiple cell and tissue types. Individually, the actions of individual growth factors have been studied. TGF-β is a member of a superfamily of >100 growth factors that include the bone morphogenic proteins. TGF-β has primary anabolic effects in musculoskeletal tissues. It stimulates mesenchymal cell proliferation, endothelial cell chemotaxis, and angiogenesis; regulates endothelial, fibroblast, and osteoblast mitogenesis, collagen synthesis, and collagenase secretion; and inhibits macrophage and lymphocyte proliferation. PDGF is found in high concentrations in α granules. PDGF is mitogenic for both mesenchymal and osteoblast cells; stimulates chemotaxis and mitogenesis in fibroblasts, glial, and smooth muscle cells; stimulates macrophage and neutrophil chemotaxis; and regulates collagen turnover. EGF stimulates angiogenesis, endothelial chemotaxis, epithelial, and mesenchymal mitogenesis; and also regulates collagen synthesis. FGF-2 promotes growth and differentiation of osteoblasts and chondrocytes, and mitogenesis of mesenchymal cells. Vascular endothelial growth factor (VEGF) promotes angiogenesis, mitogenesis of endothelial cells, and increases vessel permeability. CTGF also promotes angiogenesis, platelet adhesion, fibrosis, and cartilage regeneration. IGF has broad anabolic effects on multiple cell types1,28,29. Thus, the individual actions vary with regard to the environment in which the growth factors are placed, and the effects of these growth factors may be complementary or even inhibitory.
The advantage of using concentrated platelets is the ability to deliver an increased amount of growth factors in an autologous biologic carrier, although marked variability of growth factor content between donors and methods of platelet-rich plasma preparation has been noted30. It is assumed that these specific factors have been selected for release by platelets for their ability to initiate tissue-healing after injury.
Our model was designed to test the effects of this milieu of growth factors on a variety of normal tissue types. We expected the bioactivity of platelets to elicit a response different from normal saline solution controls within the injected tissues. The earliest time frame examined at two weeks showed a lymphocytic and monocytic inflammatory response and early new collagen fiber formation. On the basis of generally accepted models of acute tissue injury and healing, this two-week time frame would exceed the period of acute inflammation generally seen in the first week after the injury. It appears that the two-week time frame represents a period of transition to chronic inflammation and early proliferation. At six weeks, the chronic inflammatory infiltrate persisted but was decreased. Disorganized collagen formation and fibrosis with neovascularization were prominent.
We designed one arm of the study to mimic a clinical situation in which a patient may be reinjected at another time point to boost the effect of the therapy. Reinjection at six weeks with platelet-rich plasma did not appear to substantially alter the course of tissue changes, lacking a notable return of the acute inflammatory response, or noticeable increase in vascularity or scar formation. This muted response from the second injection may be related to local factors present within the tissues at this later time frame that could act to downregulate the acute inflammatory response.
Most of the effects seen histologically in this rabbit model may be construed as potentially beneficial to promote soft-tissue healing, particularly in stagnant, degenerative, nonhealing wounds. However, some potentially negative effects were also seen. A transitory early skin reaction was present in more superficial injection sites. Although platelet-rich plasma may have a beneficial effect on open wounds as in diabetic patients, injection adjacent to normal skin may create a similar response. While no studies on the human use of platelet-rich plasma have described such a skin reaction, it may be advisable to educate patients of this possibility. We surmised that these skin lesions may be related to injecting a relatively large volume of growth factors (0.5 mL of platelet gel in a 5-kg rabbit versus 2 to 3 mL of platelet gel in a human) into uninjured soft tissue.
Intra-articular injection stimulated a synovial reaction, although most clinical uses for intra-articular platelet-rich plasma involve operative procedures in which postoperative inflammation would already be present such as total joint arthroplasty or meniscal repair. This synovial reaction was generally focal and noninvasive of adjacent bone and articular cartilage, and no detrimental changes were observed in the articular cartilage.
Of greatest concern is the reaction demonstrated with intramuscular injection of platelet-rich plasma. There appears to be a substantial negative effect in normal healthy muscle with development of thrombosis and necrosis. Calcification was present at the injection site within muscle, eliciting an early inflammatory response, followed by a “cleanup” process with multinucleated giant cells. The histological pattern of calcification was not identical to that of heterotopic ossification, but may represent a similar response to muscle injury. Whether this phenomenon would have an important clinical effect in humans is unknown. Our data are consistent with the idea that exogenous calcium may exacerbate calcification of muscles because the frequency of calcified muscle lesions was greater if exogenous calcium was used to activate thrombin. However, the additional calcium chloride used to activate the thrombin may have had nothing to do with the calcification that was seen in the muscle. Activated platelet-rich plasma has a different composition of growth factors and proteolytic enzymes than platelet-rich plasma that has not been activated. Not enough calcium was present in the injected platelet-rich plasma to have caused the effect observed.
Examining the skeletal muscle calcification in greater detail, we found positive staining using alizarin red S to confirm the presence of calcium salts seen in the von Kossa-stained slides. Polarized microscopy showed these crystals to be nonbirefringent, indicating that these calcium salts were likely hydroxyapatite rather than urate or pyrophosphate. The morphologic changes witnessed in the quadriceps muscle of our rabbits and the timing of such are similar to those described in osteopontin-null mice and tumor necrosis factor receptor-null mice, both with a wild-type C57B6 background, following intramuscular injection of cardiotoxin, an inhibitor of plasma membrane Ca/Mg-ATPase31,32. Similar changes have also been demonstrated in the cardiac muscle of pigs that had undergone occlusion and reperfusion of a coronary artery to mimic acute myocardial infarct33. We surmised that injection of platelet gel into skeletal muscle would produce acute dystrophic calcification brought about by small vessel thrombosis and ischemia, followed by myonecrosis and a subsequent giant-cell response to aid in removal of the deposited calcium crystals. We further surmised that exogenous calcium would increase the likelihood of intramuscular calcification in response to ischemia.
In contrast to the findings in muscle, there were no calcified lesions in the two-week subcutaneous sites injected with platelet-rich plasma without calcium activation, whereas four of six animals injected with platelet-rich plasma and exogenous calcium had calcified lesions. This might be due to the technical difficulty of finding lesions in the large specimens used to sample the subcutaneous sites. In support of this possibility is the fact that, at six weeks, we identified a calcified site in only one of six animals injected with platelet-rich plasma with calcium. Alternatively, our data can be interpreted as indicating that calcification in subcutaneous tissues is caused by different mechanisms that require platelet-rich plasma and high local levels of exogenous calcium. There is no evidence that those mechanisms are associated with tissue necrosis similar to that seen in the muscular calcifications.
Potential weaknesses of this study include the use of the rabbit as a model. The rabbit model was chosen primarily for economical reasons, but its applicability to humans is not known. Despite the use of skin tattooing to attempt to localize sites for histological evaluation, there was the potential for sampling bias. Our preparation of the platelet gel differed slightly from that used in human clinical situations, mainly in the preparation of the activator. To gain a longer working time for the gel and to prevent it from clotting within the syringe, a higher concentration of CaCl was used (5000 U of thrombin in 30 mL versus 15 mL), and an activator to platelet-rich plasma ratio of 1:5 was used instead of the normal 1:12 used for human application. This factor would more than double the amount of calcium in the injected material and may have influenced the propensity for soft-tissue microcalcification in the first two groups. Also related specifically to the platelet-rich plasma, there was variability in platelet concentrations as noted in the table in the Appendix. Further differences specific to the function of rabbit platelets compared with human platelets are unknown. The processing and cutting of the sections in some cases appeared to create artifact or areas of clefts in the soft tissues.
Finally, this was a descriptive histological study, and no attempt was made to quantify the extent of the changes over time.
In conclusion, platelet-rich plasma is an easily obtainable autologous product that can cause a reaction in normal soft tissues in rabbits that appears histologically similar to the traditional acute “healing response” (inflammation followed by fibrosis). Transient mild skin reaction may be noted with the use of platelet-rich plasma in superficial sites at high dosages. The potential harmful effects of injections of platelet-rich plasma should be considered prior to injection into normal healthy muscle tissue.
A table showing platelet counts in serum and platelet-rich plasma and figures showing a longitudinal section of a saline solution-injected MCL, a transverse section of a platelet-rich plasma-injected MCL, normal control periosteum, and hypercellular periosteum in a platelet-rich plasma-treated animal are available with the online version of this article as a data supplement at jbjs.org.
Note: The authors thank Jerry Steinbrecher, MD, Brent Ford (MLS), ASCP, and Cynthia Henderson, HT, ASCP, for the assistance that they gave to this project.
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Disclosure: One or more of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of an aspect of this work. In addition, one or more of the authors, or his or her institution, has had a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. Also, one or more of the authors has had another relationship, or has engaged in another activity, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.