Regenerative Treatments in Sports & Orthopedic Medicine

Springer Publishing,  September 2017

CHAPTER 9: Amniotic and Umbilical Cord Products, Aplha-2 Macroglobulin, and Interleukin-1 Receptor Antagonist Protein

By Sean Colio, Marko Bodor, and  Ryan Dregalla

In this chapter we review three emerging areas in regenerative medicine for orthopedic condi- tions: amniotic products, interleukin-1 receptor antagonist protein (IRAP), and alpha-2 macro- globulin (A2M). Amniotic fluid and membranes contain numerous growth factors, cytokines, anti-inflammatory proteins, collagen, fibronectin, mesenchymal stromal cells, epithelial cells, and hyaluronic acid. These components are appealing for their utility in treating various acute and chronic musculoskeletal pathologies. IRAP is a naturally occurring analog and a com- petitor of interleukin-1 (IL-1) and binds to the interleukin-1 receptor (IL-1R) causing suppression of inflammation typically caused by IL-1. By suppressing the IL-1 inflammatory cascade, it may be possible to prevent the activation of macrophages, monocytes, and stimulation of osteoclasts that break down bone and cartilage matrix in orthopedic injuries and degenerative processes. A2M is a plasma glycoprotein with a unique ability to inhibit metalloproteinases (MMP) involved in degrading cartilage and inflammatory cytokines production. Similar to IRAP, A2M may help reduce the catabolic process in degenerative and inflammatory orthopedic conditions. Throughout this chapter we also review the current clinical evidence regarding the use of these techniques

AMNIOTIC FLUID AND TISSUE PRODUCTS

Amniotic products are derived from human amniotic fluid and amniotic membranes. The membrane forms the amniotic sac and the lining of the placenta while the fluid surrounds the fetus during pregnancy providing protec- tion and nourishment. Amniotic membranes contain numerous growth factors, cytokines, anti-inflammatory proteins, collagen, fibronec- tin, mesenchymal stromal cells, epithelial cells, and hyaluronic acid (HA) (1,2). These compo- nents convey anti-inflammatory, antimicrobial, and anti-fibroblastic properties. Amniotic fluid contains nutrients necessary for fetal development and also a chemical profile similar to that of synovial fluid with hyaluronan, lubrican, cholesterol, and cytokines (3). Amniotic tissues are also non-immunogenic, a potential source of pluripotent cells, and provide a tissue scaffold promoting wound healing and reduced scar formation (1). Stromal and epithelial cells extracted from amniotic membranes display characteristics of mesenchymal stem cells (MSC) capable of differentiating into myocytes, osteocytes, and chondrocytes (4–9). Miki et al. have described these stem cells as being non-tumerogenic on transplantation (4,6,10).

Numerous commercial forms of amniotic products are available, containing varying amounts of cryopreserved amniotic fluid, amniotic fluid-derived cells, and amniotic membrane. With respect to orthopedic applications, com- mercial amniotic products either employ the use of amniotic fluid allegedly containing a cell suspension or a micronized amniotic membrane that can be suspended in liquid and administered by injection. It is believed that some of the biological properties of amniotic tissues are retained when processed to be stored in either cryopreserved or dehydrated states. Koob et al. demonstrated that cytokine content varied significantly among amniotic membrane products, but they were able to elute the growth factors into saline and stimulate the migration of MSC both in vitro and in vivo (2).

Human amniotic fluid and tissue products are manufactured to be regulated under the U.S. Food and Drug Administration (FDA) regulation of human cells and tissues intended for implantation, transplantation, or infusion through the Center for Biologics Evaluation and Research, under Code of Federal Regulation (CFR) title 21, parts 1270 and 1271. Most manufacturers of amniotic products seek to be regulated solely under Section 361 of the Public Health Service (PHS) Act of 1944. Per the FDA, this classifica- ion allows for low-risk human cell and tissue products to enter the market and be sold with- out any safety or efficacy studies. To be solely regulated under this section, a product must sat- isfy the FDA’s definitions of being: (a) minimally manipulated, and free of any process that alters the original relevant or biological characteristics; intended for homologous use, where the tis-sue used is known by the FDA have the same basic function(s) in the recipient as in the donor; and free of viable cells in the case of an allograft, as the product may not rely on metabolic activ-ity of the If a human cell or tissue prod-uct does not meet all of the criteria mentioned, then it will be regulated by the FDA as a medical device (class II or III), a drug, or under Section 351 of the PHS Act, which parallels the approval process for a drug but for products comprised exclusively of human cells and tissues. In each of these instances, premarket approval of the product by the FDA is required, which becomes intensely more time-consuming and financially burdensome. In recent history, many amniotic products intended for orthopedic applica-tions have been disqualified from the privilege of being solely regulated under Section 361 for three primary reasons: (a) the product description includes claims of viable (allogeneic) cells in the product that are ancillary to the product’s function; (b) the morselization of a membrane alters the original relevant characteristics of the product from a mechanical and dimensional perspective and therefore is more than minimally manipulated; and (c) any homogenized or morselized form of amniotic membranes labeled for orthopedic use (injectable) are not for homologous use as it does not “replace or supplement dam- aged or inadequate integumental tissue.” Hence, many amniotic products intended for orthopedic applications have been reassigned to a new classification; most have received notification that the product will be regulated under Section 351 of the PHS Act, and are currently undergoing the necessary studies to demonstrate product safety and efficacy to remain in the marketplace.

Amnion-derived products have been utilized in the treatment of corneal injuries, skin wounds, burns, and leg ulcers (2,11–20). One of the earliest publications on their use for orthopedic conditions was in 1938 in which bovine amniotic fluid concentrate was used within the joints of 68 patients (21). In this study, Shimberg et al. recognized that the amniotic fluid mechanism of action was likely both mechanical and biological despite the limited understanding at the time of the composition of the amniotic concentrate.

The interpretation of their results was that there was a beneficial mechanical distention of the joint, a defense reaction in the intra-articular tissues due to fibrin formation, stim-ulation of a repair process, and improvement of intraarticular fluid viscosity by preventing adhesions (21).

Currently, there are numerous in vitro and in vivo studies evaluating the use of amniotic compounds in treating cartilage, tendon, ligament, and fascial injuries (22–31) The majority of these studies use animal models and at this time only three human studies have been published. Philip et al. and Kueckelhaus et al. demonstrated improved mechanical properties in Achilles tendons in rats after treatment with amniotic solutions (24,32). Coban et al. found no benefit in treating ruptured Achilles tendons in rats with amniotic fluid and membrane (33). Other animal studies have looked at the effect of amniotic compounds on digital flexor tendon repair, demonstrating a decreased incidence of adhesion formation (34–36). There are no human studies evaluating the effects of amniotic compounds on human tendon and ligament healing; however, there are two studies on plantar fasciitis.

Zelen et al. published an industry-sponsored, prospective, randomized, single-center clinical trial of 45 patients with chronic refractory plantar fasciitis (37).They were randomized into three groups to receive an anesthetic injection of 2 mL 0.5% marcaine, followed by either 1.25 mL saline (controls), 0.5 mL micronized dehydrated amniotic membrane (MDAM), or 1.25 mL MDAM. Follow-up visits were scheduled weekly for 6 weeks, and a final study visit was scheduled at 8 weeks post injection. Three outcome measure scales were used during the evaluation: the American Orthopaedic Foot and Ankle Society (AOFAS) hindfoot scale, the Wong– Baker FACES Pain Rating Scale, and the SF-36v2 standard health survey. Baseline AOFAS scores were 54.4 ± 17.7, 41.3 ± 4.5, and 41.0 ± 7.7 inn the control, 0.5 mL MDAM, and 1.25 mL MDAM groups, respectively. The scores rose in all groups throughout the study, with final scores at 8 weeks being 70.0 ± 9.6, 92.9 ± 8.7, 94.3 ± 5.6 in the control, 0.5 mL in the MDAM, and 1.25 mL in the MDAM groups respectively. Additionally, the SF36v2 mental and physical component scores rose and the FACES scores dropped for both MDAM groups more so than compared with the controls. Baseline scores on the SF36v2 physical component were 41.4 ± 6.1 for the con- trol, 36.0 ± 5.9 for the 0.5 mL MDAM group, and 37.0 ± 3.8 for the 1.25 mL MDAM group and rose to 43.6 ± 5.6 for the control, 55.9 ± 3.5 for the 0.5 mL MDAM group, and 57.3 ± 2.6 for the 1.25 mL MDAM group. The authors reported no adverse events in either group. Due to the short 8-week follow-up, the editor of the journal termed this a feasibility study and noted that the authors were conducting a longer term follow-up study.

Hanselman et al. published an industry- sponsored–double-blind randomized, controlled, single-center pilot study of 24 patients with chronic plantar fasciitis (38). They randomized two groups: (a) a control group receiving corticosteroid (1 mL of 40 mg/mL Depo Medrol, 4 mL bupivacaine 0.5%) and (b) a study group receiving cryopreserved human amniotic membrane (1 mL cryo- preserved human amniotic membrane [CHAM], 4 mL bupivacaine 0.5%). The groups were evaluated over three clinic visits: an initial visit at the time of the injection, a 6-week follow-up, and a 12-week follow-up. The primary outcome measure was the Foot Health Status Questionnaire (FHSQ) with higher scores implying reduced pain and improved function. The secondary outcome measure was the Visual Analog Scale (VAS) and the patient’s verbal self-report of percent improvement. The average FHSQ scores at 6 weeks were: foot pain 24.6 in the corticosteroid group and 18.8 in the CHAM group, foot function 8.3 in the corticosteroid group and −2.1 in the CHAM group, and physi- cal activity 14.8 in the corticosteroid group and 5.6 in the CHAM group. After the 6-week follow-up, when most of the FHSQ scores were higher in the corticosteroid group versus the CHAM group, the study groups were offered a second injection. Each group then underwent the same injection proto- col with the same drug that corresponded to their initial injection, which was again blinded to both the investigator and the patient. They were then reevaluated twice more at 12-week and 18-week follow-up visits. At 18 weeks the FHSQ scores were as follows: foot pain 32.5 in the corticosteroid group and 66.3 in the CHAM group, foot function 33.3 in the corticosteroid group and 31.3 in the CHAM group, and physical activity 31.5 in the corticosteroid group and 33.3 in the CHAM group. Thus, at 18 weeks most of the FSHQ scores were higher in the CHAM group versus the corticosteroid group, a reversal of the findings seen at 6 weeks. The VAS scores decreased from baseline, with the single injection subset being −12.6 for the corticosteroid group and −17.8 for the CHAM group at 12 weeks, and the double injection subset being −25.7 for the corticosteroid group and −37.3 for the CHAM group at 18 weeks. The authors reported no adverse side effects from either injection. The editor of the journal opined that given the short follow-up period, the study showed that only CHAM was safe in the short term and about as equally effective as a corticosteroid injection but at a higher cost.

There are many cell culture and animal studies investigating the use of amniotic membranes for cartilage pathology (22,23,25,39–42). However, human studies are limited with only one published open-label, prospective feasibility study. In this study, Vines et al. selected six patients with Kellgren–Lawrence knee osteoarthritis grades of 3 and 4 (43). They administered a single intra-articular injection containing cryopreserved, particulate human amnion and amniotic fluid cells with follow-up of the patients at 1 and 2 weeks and at 3, 6, and 12 months post-treatment. Outcome measures included the Knee Injury and Osteoarthritis Outcome Score (KOOS), International Knee Documentation Committee scale (IKDC), and a single assessment numeric evaluation pain scale (SANE). The KOOS outcome score improved from a baseline of 43.35 to 70.23 by the 1-year time point. The IKDC assessment improved from an average score of 41.7 at baseline to 63.4 at 6 months and to 64.4 at 1 year. SANE scores improved from an average of 51.25 at baseline to 87.3 at 6 months and 85.8 at 1 year. The authors determined that statistical analysis was not appropriate for their data. The authors reported no adverse events other than a transient increase in knee pain that resolved at a 2-week follow-up (43).

In summary, although there are a number of animal and cell culture studies regarding the use of amniotic products for orthopedic conditions, the current clinical evidence for humans is limited to the one feasibility study for knee arthritis and the two small randomized controlled trials for plantar fasciitis. Additional clinical trials for more types of musculoskeletal pathologies along with larger sample sizes and longer fol- low-ups are needed. It is interesting to note that the published utilization of amniotic products in orthopedics dates as far back as 1938, preceding many other areas of regenerative medicine. Amniotic membranes are a potential source of viable pluripotent MSC in the fresh state and growth factors in the cryopreserved state, yet whether these products provide a benefit to MSC conditions remains unknown (1). With ongoing clinical research, the beneficial effects of amniotic products hypothesized by Shimberg et al. in 1938 may eventually be realized.

UMBILICAL CORD BLOOD

Umbilical cord blood (UCB) is blood left in the umbilical cord after childbirth. It consists of red and white blood cells, plasma, and platelets. It is also a rich source of hematopoietic and mesenchymal stromal cells. Collection of cord blood is typcally done by cannulating the vein of the umbilical cord after it is severed. The amount of UBC from a donor differs but can range from 75 to 150 mL on an average (44). Processing then varies greatly depending on the tissue center or manufacturer. The UCB collected is then cryopreserved (45).

Clinical experience with UCB since its first use in the late 1980s is mostly with hematopoietic reconstitution; however, increased understand- ing of the cell populations has expanded its potential in cellular immunotherapies for therapeutic use against malignancies (46). UCB-derived MSC are in a more primitive state without antigen-producing cells, rendering them invisible to the hosts immune system permitting allogenic cell therapy (47). Animal and human studies are currently underway in spinal cord and brain injury applications, cerebral palsy, lung disease, kidney injury, juvenile diabetes, and autoimmune applications (48).

Recently, a processing methodology (Pro-genokine®, Smart-Surgical Inc. dba Burst Biologics, Boise, Idaho) was developed to preserve the integrity of UCB cells and protecting cell viability. This processing method avoids using toxic media, including dimethyl sulfoxide (DMSO), to protect cell viability (49–51). As a result, these cells can self-renew after a freeze–thaw cycle and avoid apoptosis. One of the first commercially available products using this method is an injectable/fluid allograft stem cell product derived from UCB (BioBurst Rejuv®, Burst Biologics, Boise, ID). It is regulated as a minimally manipulated tissue under CFR 1271 for homologous use only. Currently there are only unpublished anecdotal reports with clinical studies in process (52).

INTERLEUKIN-1 RECEPTOR ANTAGONIST PROTEIN

Inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and IL-1 have been implicated in the pathogenesis of osteoarthritis due to their catabolic mechanisms on cartilage. These factors mediate the function of a variety of cells, including phenotype shifts in macrophages that are major players in the progression of osteoarthritis, (53,54) express tissue- damaging metalloproteases (55) and contribute to disease progression through continued expression of proinflammatory cytokines (56,57). The discovery of competitive antagonists of these cytokines has greatly evolved the treatment of rheumatoid arthritis and is now offering insight into new techniques at halting the degenerative process of osteoarthritis. IRAP, also known as IL-1 receptor antagonist (IL-1Ra), is a naturally occur- ring analog and competitor of IL-1 and binds to the IL-1R receptor, with an affinity for type I and II receptors (58,59). When IL-1 binds to an IL-1R receptor, a cascade of inflammation is triggered, including activation of macrophages, monocytes, and stimulation of osteoclasts to break down bone and cartilage matrix (58–61). However, when IRAP binds to an IL-1R receptor, inflammation is suppressed. One hypothesis is that an appropriate balance of IRAP to IL-1 leads to a healthy equilibrium between anabolic and catabolic processes in joints and muscles (62–66). If that equilibrium is unbalanced toward excess IL-1 or insufficient IRAP, then a propensity toward the destruction of cartilage, muscles, and other joint structures ensues (62–66). Thus, there has been an interest in developing methods at isolating and producing IRAP.

In 1990, Seckinger et al. described natural and recombinant human IL-1 receptor antagonists blocking the effects of IL-1 on bone resorption and prostaglandin production (67). Since that time, recombinant versions of IRAP have been developed and tested with Jiang et al. publishing a multicenter, double-blind, dose-ranging, randomized, placebo-controlled study evaluating its utility for rheumatoid arthritis (RA) patients (68). They concluded that their systemic recombinant human IL-1 receptor antagonist reduced radio- logic progression of RA. The recombinant version of IRAP was branded as anakinra and based on its effects on RA, Chevalier et al. investigated its use in osteoarthritis (OA). They published a safety study on the intra-articular injection of anakinra as an earlier use in RA was systemic administration (69). They reported that intra-articular injection of IRAP in patients with knee OA was well tolerated and did not induce any acute inflammatory reactions and also noted an improvement in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) scores until month 3. Chevalier et al. followed up on this study with a multicenter, double-blind, placebo-controlled study and randomized 2:1:2 to receive a single intra-articular injection of placebo, anakinra 50 mg, or anakinra 150 mg in their symptomatic knee (70). The study groups were followed for 12 weeks with the primary end point being the change in the WOMAC score from baseline to week 4. Out of 160 (94%) patients who completed the study, the mean improvements from baseline to week 4 in WOMAC scores were not statistically different among groups. They reported no adverse events.

Further studies on anakinra for intraarticular inflammatory processes have been more positive. Brown et al. retrospectively reviewed six patients (three female and three male), ranging in age from 17 to 50 years, who underwent injection of intraarticular anakinra, 200 mg, for persistent effusions of their postoperative knee (71).They reported that after intra-articular anakinra, 66% had improvement in knee arc of motion (15–30°) and pain, five of six (83%) had improvement in swelling and all of these patients were able to return to sports. Kraus et al. evaluated 11 patients with acute anterior cruciate ligament (ACL) tear confirmed by MRI and randomized them to receive a single intra- articular injection of anakinra 150 mg or equal volume of saline placebo 2 weeks after injury and before surgical ACL reconstruction (72). They concluded that anakinra decreased pain and improved function (activities of daily living [ADL], sports function, and quality of life [QOL]) and exceeded the minimal perceptible clinical improvement in ADL for both the WOMAC and KOOS.

Arend et al. described IRAP production using surface-bound immunoglobulin G, lipopolysac- charide, phorbol myristate acetate, IL-1, and TNF- α to induce IRAP production by isolated human monocytes (73–77). Based on this understanding, Meijer et al. published a method to concentrate peripheral blood leukocytes from venous blood through centrifugation and then incubating them over glass beds to induce IRAP production de novo (78). This product was named as autologous-conditioned serum (ACS) and branded as Orthokine®. This process also produces a variety of likely beneficial growth factors and cytokines within ACS, including vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) AB, hepatocyte growth factor (HGF), Insulin-like growth factor 1 (IGF-1), and trans- forming growth factor (TGF)-β (60,64).

There are several uncontrolled studies evaluating the use of multiple ACS injections for knee and hip OA all showing improvement in WOMAC scores (79–82). Two randomized placebo-controlled studies compared ACS with saline showed statistically significant improvements in KOOS and VAS scores (83,84). Yang et al. reported that ACS provided no greater improvement in WOMAC scores compared with saline and they concluded that because their primary efficacy objective was not met, they could not yet recommend the use of ACS for the treatment of OA (83). They also reported two adverse events, septic arthritis and an inflammatory reaction of the knee joint. Baltzer et al. included a HA group in their randomized-controlled trial (RCT) and concluded that the effects of ACS were superior to those of HA and saline for all outcome measures and time points, and improvements were clinically relevant (84). They also reported that the frequency of adverse events was highest in the HA group.

Intra-articular application of ACS has been published as an adjunct to ACL reconstruction. Darabos et al. published level 1 therapeutic RCT study demonstrating that ACS reduced bone tunnel enlargement with four injections after ACL reconstruction (85). They reported that the enlargement in the ACS group at 6 months was 8%, 12 months being 13%; in the saline group at 6 months it was 31%, 12 months being 38%. They noted that WOMAC and IKDC scores were consistently better in patients treated with ACS than saline. Darabos followed up with a study comparing double-bundle anterior cruciate ligament reconstruction with and without ACS how- ever, the article was retracted at the request of the corresponding author due to inconsistencies in the described method and incorrect reporting of conflicts of interest (86).

ACS use for muscle injuries has been described by Wright-Carpenter et al.’s experimental study in which mice received blunt trauma to their gastrocnemius muscle (87). They divided the study group in half, with one group receiving saline and the other receiving ACS at 2, 24, and 48 hours after the injury. Histology results showed that satellite cell activation at 30 to 48 hours post injury was accelerated and the diameter of the regenerating myofibers was increased compared with the controls within the first week after injury (87). Based on these results Wright-Carpenter et al. performed a pilot study of 18 athletes (soccer, basketball, ice hockey) with second-degree muscle strains diagnosed by MRI (hamstring, adductor, iliopsoas, gluteus, abdominal oblique, gastrocnemius, rectus fem- oris) (88). ACS injections started 2 days after the diagnosis and were administered every second day with the mean number of treatments per patient being 5.4. The control group in the study was a retrospective analysis of 11 patients treated with Actovegin®/Traumeel® therapy. The authors reported an average time to recovery of 16.6 ± 0.9 days with follow-up MRI scans taken at 14 to 16 days after injury showing near complete regression of the findings in the first scan concerning edema, bleeding into the mus- cle, and restitution of the muscle structure. In the control group they reported an average time to recovery of 22.3 ± 1.2 days with follow-up MRI scans taken at 14 to 16 days after injury showing only a mild regression of the findings in the first scan concerning edema and bleeding into the muscle. The authors reported no local or systemic side effects in either group.

Data regarding ACS use in tendon injuries is limited to two in vitro and in vivo studies. Both studies applied ACS to damaged rat Achilles tendons (89,90). Majewski et al. reported that ACS-treated tendons were thicker, with more type I collagen, and demonstrated an accelerated recovery of tendon stiffness and histologic maturity of the repair tissue (89). However, they reported that there were no differences in the maximum load to failure between the ACS and the untreated groups up to week 8. Heisterbach et al. noted that the expression of growth factors basic fibroblastic growth factor (bFGF), bone morphogenetic proteins (BMPs)-12 and TGF-β1 in the ACS-treated tendons was significantly greater than controls but VEGF was not affected (90). Both studies concluded that ACS has the potential to improve Achilles tendon healing.

ACS has been studied as a potential treatment for cervical and lumbar radiculopathy (91,92). Becker et al. published a single center, prospective, double-blind, reference-controlled, investigator-initiated study comparing 32 patients treated with epidural perineural ACS injections to 27 patients treated with 5 mg triamcinolone and 25 patients with 10 mg triamcinolone (91). ACS injections were performed once per week for consecutive weeks and followed for 6 months. The primary outcome measure was the VAS and the Oswestry Disability Index (ODI) was the secondary end point of the study. The authors reported that from week 12 to the final evaluation at week 22, the ACS group had lower VAS scores compared with both triamcinolone groups, but statistical significance was observed only at week 22 in direct comparison with the triamcinolone 5 mg group. They noted that ODI scores were already reduced in all treatment groups at week 6 and although the ACS group showed a greater reduction in ODI scores at week 10, at the final 6-month evaluation ODI scores were similar in all treatment groups.

Goni et al. published a prospective randomized pilot study on 40 patients with unilateral cervical radiculopathy equally divided into two groups: one receiving 2.5 to 3 mL of ACS and the other receiving 2.5 to 3 mL methylprednisolone both under fluoroscopic guidance into the neural foramen (92). They were followed for 6 months using the VAS for neck pain, neck disability index (NDI), and Short Form of Health Survey-12 (SF- 12). They reported that the ACS group showed a 73.24% improvement in VAS scores over the mean baseline VAS at the end of 6 months. The methylprednisolone group showed a 58.54% improvement in VAS scores over baseline at the end of 6 months. They reported a decrease in the NDI in both groups with the percentage decrease in the ACS group being 74.47% com- pared with 52.80% in the methylprednisolone group. The SF-12 scores were divided into the Physical Health Component Score-12 (PCS-

12) and Mental Health Component Score-12 (MCS-12). The mean PCS-12 score increased by 79.45% in the ACS group while the methyl- prednisolone group showed a 57.32% increase during the same duration of follow-up. In the ACS group, MCS-12 scores improved by 30.09% while the methylprednisolone group increased by 16.16% at 6-month follow-up. In both groups they reported similar numbers of immediate (syncope, dizziness, sweating, tachycardia) and delayed complications (neck stiffness).

Based on early studies regarding the utility of IRAP in RA, further investigation regarding its ability to treat OA has been a logical outgrowth. However, the studies for both recombinant IRAP and ACS in knee OA are very limited compared with other areas of regenerative medicine. For other orthopedic conditions, both recombinant IRAP and ACS need larger, controlled studies with longer-term follow-up. ACS and IRAP both appear safe with potential applications to many other inflammatory conditions.

ALPHA 2 MACROGLOBULIN

The Inflammatory cytokines TNF-α, IL-1b, and IL-6 are major contributors to both intra- articular and extra-articular joint pathologies and pain (93). These cytokines in turn enhance the inflammatory cascade by producing more pro-inflammatory agents, including enzymes of the MMP family. These MMPs target and degrade a wide array of extracellular matrices, which can result in the deterioration of car- tilage, bone, tendon and ligaments (94,95). Furthermore, MMP activity releases catabolic byproducts, which induce the production of more inflammatory cytokines (96–98). A2M is a plasma glycoprotein with a unique ability to inhibit all endoproteases and more specifi- cally matrix MMP (99). Because of this effect, A2M may potentially slow cartilage damage by neutralizing cartilage catabolic enzymes. Wang et al. published their results on an ex vivo study showing that A2M decreased cartilage catabolism by inhibiting the protease activity of ADAMTS-5 and metalloproteinases (99).

The Autologous Protease Inhibitor Concentrate (APIC-CF) System is marketed as a method to concentrate A2M from 45 mL venous blood via centrifugation and ultrafiltration with a tangential flow filter (93). The APIC-CF system received the FDA approval for an Investigational New Drug (IND) Clinical Trial for the treatment of mild to moderate OA in July 2014. There are a few clinical studies investigating the use of A2M. In Wang et al.’s publication they used a rat model of anterior cruciate ligament transection-induced OA and found that supplemental intra-articular injection of A2M reduced the concentration of MMP-13 in synovial fluid, had a favorable effect on OA-related gene expression, and attenuated OA progression (99). Smith et al. published their findings of a spe- cific cartilage degradation product, fibronectin- aggrecan complex (FAC), in the epidural space as a reliable predictor of response to the lumbar epidural steroid injection in patients with radicu- lopathy (100) Scuderi et al. reported in a poster presentation that patients who are FAC+ within the intervertebral disc are more likely to demon- strate clinical improvement in both VAS and ODI scores following intradiscal autologous A2M injec- tion (101). They suggested that A2M may be an efficacious biologic treatment in discogenic pain and that the FAC may be an important biomarker in patient selection for this treatment. More clin- ical studies are needed to understand A2M’s utility in degenerative and inflammatory orthopedic conditions.

CONCLUSION

The majority of studies investigating the use of amniotic products, IRAP, and A2M are limited to in vitro and animal studies with few human clinical trials. Additional randomized clinical trials including other musculoskeletal pathol- ogies along with larger sample sizes and lon- ger term follow-ups are needed to understand their future potential. Furthermore, most of the trials have compared their efficacy versus corticosteroids and saline rather than other orthobiologics. Corticosteroids have known dose-dependent side effects with risks, includ- ing osteoporosis, osteonecrosis, deterioration of articular cartilage and tendon or ligament weakening or rupture leading to minimizing their chronic usage and in some cases avoiding them altogether. Intra-articular saline injections have been shown to yield a statistically and clinically meaningful improvement in knee OA symptoms for up to 6 months, which exceed a placebo effect (102). Therefore, a more logical compar- ison would be evaluating these products versus other regenerative medicine techniques, such as HA, platelet-rich plasma, or MSC. Nonetheless, improvements have been made in the produc- tion and isolation of these products allowing this research to advance, a great stride forward when compared with the early use of bovine amniotic fluid in the joints of patients described in 1938.

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