- Email: [email protected]
A short report on the effect of decreased incubation time on the architectural profile of autologous conditioned serum (ACS)
Cytokine xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
A short report on the effect of decreased incubation time on the architectural profile of autologous conditioned serum (ACS) Angelique Barreto, MD Memorial Clinical Research, Oklahoma City, OK, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Autologous conditioned serum Osteoarthritis IL-1-Ra IL-1 IRAP
If present in high enough concentrations, IL-1-Ra has the potential to inhibit Interleukin-1, the chief offender that promotes the pro-inflammatory cascade causing pain, swelling and joint dysfunction associated with osteoarthritis (OA). IL-1-Ra and growth factor levels were quantified from whole blood in this retrospective chart review investigation (n = 20) using Zero and 15 min incubation times respectively. The hypothesis that this process can significantly (p < 0.0001) increase levels of IL-1-Ra was confirmed. Mean Arthrokinex™ induced IL1-Ra levels reached a concentration of 13,288 pg/mL and 12,809 pg/mL compared to 518 pg/mL at baseline, representing a 26-fold increase. Post conditioning levels of pro-inflammatories IL-1β, IL-6 and TNF α were not changed to any significant degree. The Arthrokinex™ blood conditioning process induces adequate levels of IL-1Ra to alter the IL-1-Ra: IL-1β ratio and mitigate the inflammatory cascade, while increasing growth factors PDGF and TGF respectively.
1. Introduction Traditionally, the clinical characteristics of osteoarthritis (OA) were thought to be part of the normal aging process and often classified incorrectly as degenerative joint disease. More recent studies have revealed the complex inflammatory mechanisms that cause articular cartilage destruction, sub-chondral bone thickening and synovitis. Despite these advances, the multi-factorial pathogenesis of OA, estimated to affect 27 million people in the US [1], is still poorly understood. Currently recommended pharmacologic interventions are aimed solely at symptomatic control forcing many patients to undergo surgery. Regenerative therapies such as Platelet Rich Plasma (PRP) injections are on the rise despite conflicting evidence. Given the limited effectiveness of analgesic drugs, NSAIDs and intra-articular corticosteroid injections, as well as the significant financial burden associated with treating an aging population, development of a chondroprotective drug is needed to augment joint function and reduce pain in the 67 million people OA is estimated to affect by 2030. Interleukin-1 (IL-1) is a pro-inflammatory cytokine that affects nearly every cell type [2]. Overwhelming evidence from in vitro and in vivo studies implicate IL-1β as the key mediator of the inflammatory process in OA [3]. Chondrocytes embedded within articular cartilage matrix are influenced by a variety of cytokines and growth factors that facilitate destruction and synthesis of new matrix proteins [4]. Under conditions of disease, dysregulation of this delicate balance, chiefly between IL-1β and its naturally occurring inhibitor, Interleukin-1
Receptor antagonist (IL-1-Ra), leads to matrix degradation. The Arthrokinex™ formulation has proven to consistently induce autologous conditioned serum(ACS) containing extremely high levels of the antiinflammatory cytokine, IL-1-Ra, as well as growth factors that promote cartilage regeneration [5]. Previously, a 30 min incubation time was reported as necessary to induce a 32-fold increase in IL-1-Ra from baseline. The purpose of this investigation is to determine if decreasing the incubation time has an effect on the architectural profile of ACS. It is hypothesized that decreasing the incubation time will decrease levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and will produce significant elevations of anti-inflammatory cytokine levels (IL-1-Ra and IL-10). 2. Methods 2.1. Study design Data collection was conducted in two separate locations in Oklahoma City, OK, USA and one location in Austin, TX, USA between March 2014 and July 2016. All aspects of this retrospective chart review were reviewed and approved by IntegReview Institutional Review Board (IRB) as being considered exempt from requiring IRB approval as it met all requirements outlined in 45 CFR 46.101(b)(4), specifically (1) the research involved only the collection or study of preexisting data, documents, records, pathologic specimens or diagnostic specimens and (2) the information was recorded in such a manner that
E-mail address: [email protected]. http://dx.doi.org/10.1016/j.cyto.2017.03.019 Received 26 February 2017; Accepted 31 March 2017 1043-4666/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Barreto, A., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.03.019
Cytokine xxx (xxxx) xxx–xxx
A. Barreto
the subjects could not be identified directly or through identifiers linked to the subjects.
Table 1 Cytokine and growth factor levels of pre and post ACS conditioned serum. Serum
Arthrokinex™ Induced ACS
N = 20
No incubation time N = 10
15 min incubation time N = 10
518
13,288a
12,809a
< 3.9 < 7.8 < 15.6
< 3.9 < 7.8 < 15.6
< 3.9 < 7.8 < 15.6
< 3.1 25,892 25,226 96
< 3.1 28,163a 52,161a 94
< 3.1 32,096a 62,101a 106
2.2. Participants A total of 20 patients with symptomatic KOA met the American College of Rheumatology (ACR) inclusion criteria for analysis. According to the ACR criteria, patients with at least three of the six primary clinical features were diagnosed with KOA. These symptoms include age > 50, morning stiffness < 30 min duration, crepitus on active range of motion, tenderness of the bony margins of the joint, bony enlargement noted on examination and a lack of palpable warmth of the synovium. Exclusion criteria included: patient charts of those in generally poor health; drug dependent (chronic opioid use, alcohol, etc.); undergone surgery or treatment of the affected joint within the last 3 months; systemic disease of the musculoskeletal system; bone cancer; metastasis or tumor-like lesions in the immediate proximity to the treated joint; fracture in the last 3 months; acute bacterial infection; blood clotting disorders; continuous corticoid or NSAID therapy due to other diseases. Informed consent was obtained from each participant and all work was performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki, 1975 revised 2008).
IL-1-Ra (pg/ mL) IL-1β (pg/mL) IL-10 (pg/mL) TNF-α (pg/ mL) IL-6 (pg/mL) TGF (pg/mL) PDGF (pg/mL) IGF-1 (ng/mL)
a Statistically significant (p value < 0.0001) elevation when compared to pre conditioned serum.
2.5. Statistical analysis SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The non-parametric Wilcoxon Signed Rank test was performed to analyze the statistical difference between baseline and post-processing cytokine levels. All results shown are the mean ± SEM of two or more experiments.
2.3. Processing of whole blood Using aseptic techniques, 60 mL venous whole blood of twenty (20) participants was harvested into a sterile 60 mL syringe containing 3 mL of anticoagulant citrate dextrose (ACD) solution and centrifuged (3200 rpm, 15 min). The resultant Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP) were extracted and the remaining layer containing erythrocytes, was discarded. Both the PRP and PPP were transferred to a specialized, closed-system, centrifuge tube containing medical grade concentrator beads and mixed. Ten samples were incubated for 15 min and 10 samples were incubated for zero minutes. After the reduced incubation period, centrifuge filtration (2000 rpm, 3 1/2 min) through a sterile 0.45 µm filter was completed and the resulting sterile filtrate obtained. The ACS could be used immediately for intra-articular injection or stored at −20 degrees Celsius for future use.
3. Results and discussion The purpose of this retrospective analysis was to test the hypothesis that pro-inflammatory cytokines would decrease, extremely high levels of IL-1-Ra would be maintained and growth factors (IGF-1, TGF and PDGF) would increase after reducing the incubation time of the formulation. This hypothesis was proven correct. All pro-inflammatory cytokine levels read below the minimum value at each incubation time (Table 1). At the previous incubation time of 30 min, pro-inflammatory cytokine levels of post-Arthrokinex™ were higher than pre-conditioned serum. IL-1-Ra levels at these shorter incubation times were similar to serum conditioned at the original 30 min [5]. Additionally, PDGF and TGF levels were significantly increased following the Arthrokinex™ formulation at each incubation time. The presence of these growth factors contained in ACS supports the hypothesis that IA injections of ACS may reverse the disease process associated with OA as growth factors have been shown to stimulate chondrocyte proliferation and augment articular cartilage metabolism. At increased concentrations, IL-1-Ra has the potential to limit the destructive inflammatory intra-articular actions of IL-1β. IL-1-Ra concentrations must exceed IL-1β levels by 10–100 fold to competitively inhibit the interaction of IL-1β with its receptors. On average, knees affected with OA will contain approximately 34 pg of IL-1β [6]. This novel method provided de novo levels of IL-1-Ra that exceeded the 100:1 threshold in each of the 10 serum samples incubated at 15 min and each of the 10 serum samples incubated for zero minutes. Given the significantly increased levels of IL-1-Ra incubated for 15 min (12,809 pg/mL), the significantly increased levels of IL-1-Ra incubated at zero minutes (13,288 pg/mL), and the ratio of the Arthrokinex™ induced IL-1-Ra to the pro-inflammatory cytokine (IL-1β), it is reasonable to conclude that autologous conditioned serum(at each incubation time) can consistently antagonize IL-1β receptor binding. While at first glance this approach may appear similar to other commercially available products, this process has several important modifications. Initially, this novel formulation was designed to incubate for 24 h. However, after several attempts, it was discovered that the same levels of IL-1-Ra could be induced after the PRP/PPP mixture was in contact with the beads for only 30 min. Currently, it is demonstrated that 15 min and even zero minutes of incubation time are sufficient to
2.4. Biomarker assays The primary outcome of measuring IL-1-Ra (pre- and post-conditioning) was achieved by using the highly sensitive, commercially available quantitative sandwich enzyme-linked immunoassay technique (R & D Systems, Quantikine ELISA; Minneapolis, MN, USA). This kit, when run in accordance with standard Quantikine protocols, is extremely sensitive (minimum detectable dose ranged from 2.2 to 18.3 pg/mL), specific (no significant cross-reactivity or interference was observed), precise (intra- and inter-assay CVs were 3.7% and 6.7%) and linear (all diluted samples fell within the dynamic range of the assay). Resulting concentrations were calculated by subtracting the average zero standard optical density and log transforming IL-1-Ra concentrations versus the log of the optic density on a linear scale, and the best fit line determined by regression analysis. IL-1-Ra concentrations were only accepted if the standard curve correlation coefficient (r) reached 0.99 and the CV of each sample was under 20%. Additionally, serum levels (pre- and post-conditioning) of proinflammatory (TNF α, IL-1β, IL-6) cytokines, anti-inflammatory cytokine (IL-10) and growth factors (insulin like growth factor 1, platelet derived growth factor and transforming growth factor) were also measured using ELISA. All kits reported comparable sensitivity, specificity, precision and linearity as described above and were run in accordance with standard Quantikine protocols. 2
Cytokine xxx (xxxx) xxx–xxx
A. Barreto
clinical trials are needed to compare the effects of Arthrokinex™ (ACS) to other currently accepted therapeutic options. Meanwhile, this shorter incubation time offers an alternative, on-site, point of service treatment option for a population with an increasing burden of OA.
produce high quality ACS. Moreover the shorter incubation time decreased the pro-inflammatory cytokine profile and maintained IL-1Ra levels. Clinically this would translate into continued pain relief and less chance of a post IA injection flare. Additionally, Arthrokinex™ does not introduce any additional chemicals and utilizes a closed loop system to avoid contamination. The lack of effective treatment options for OA continues to present a challenge for patients and providers. Acetaminophen is the first line oral option available to treat mild to moderate knee OA [7] even though evidence suggests that NSAIDs have superior analgesic effects. NSAIDs are recommended for moderate to severe OA but should be used judiciously due to the well-known gastro-intestinal, renal and cardiotoxic effects [8] including stroke and myocardial infarction. The Osteoarthritis Society International (OARSI) [7] also recommends the administration of short term IA corticosteroid injections; however, serious side effects of steroid [9] injections should be considered. The chronic nature of OA and availability of short term treatment options highlights the need for a chondroprotective agent that can modify the disease process at the molecular level. Joint injections of serum rich in IL-1-Ra and growth factors, induced by this blood conditioning process, has the potential to shift the intra-articular conditions to a more favorable cytokine profile. Furthermore, this process is relatively inexpensive with an expected favorable safety profile given the biotherapeutic approach. Several clinical trials highlight other potential applications of the Arthrokinex™ process beyond the treatment of OA. Clinical improvements have been reported in muscle and ligament injury [10] and spinal degenerative disk disease [13]. Darabos et al. [11,12] demonstrated increased levels of IL-1β in the synovial fluid of 19 out of 20 knees following ACL reconstructive surgery, and resultant reduction in post surgery complications following administration of IL-1-Ra IA injections. Mitigating post-surgical inflammation could be accomplished by administering Arthrokinex™ injections. Given the overwhelming data describing many different clinical approaches of IL-1Ra, the potential of autologous conditioned serum is broad. It is important that a notable limitation due to the short half-life of the serum exists, and is overcome in large part by the administration of closely spaced IA injections of stored serum in the Arthrokinex™ clinical protocol. It was recently demonstrated that intra-articular injections of Arthrokinex™ (ACS) provided significant clinical benefits in a clinical trial involving 100 patients with symptomatic KOA [14]. Additional
References [1] C.G. Helmick, et al., Estimates of the prevalence of arthritis and other rheumatic conditions in the US, Arthritis Rheum. 58 (1) (2008) 15–25, http://dx.doi.org/10. 1002/art.23177. [2] C. Dinarello, Interleukin-1, Cytokine Growth Factor Rev. 8 (4) (1997) 253–265, http://dx.doi.org/10.1016/S1359-6101(97)00023-3. [3] M. Goldring, Osteoarthritis and cartilage: the role of cytokines, Curr. Rheumatol. Rep. 2 (6) (2000) 459–465, http://dx.doi.org/10.1007/s11926-000-0021-y. [4] L.C. Dijkgraaf, L.G. de Bont, G. Boering, R.S. Liem, Normal cartilage structure, biochemistry, and metabolism: a review of the literature, J. Oral Maxillofac. Surg. 53 (8) (1995) 924–929, http://dx.doi.org/10.1016/0278-2391(95)90283-X. [5] A. Barreto, et al., A method to induce Interleukin 1 receptor antagonist from autologous whole blood, Cytokine 81 (2016) 137–141, http://dx.doi.org/10.1016/ j.cyto.2016.03.008. [6] G.S. Firestein, J.M. Alvaro-Gracia, R. Maki, Quantitative analysis of cytokine gene expression in rheumatoid arthritis, J. Immunol. 144 (9) (1990) 3347–3353, http:// dx.doi.org/10.1008/s13567-028. [7] W. Zhang, et al., OARSI recommendations for the management of hip and knee osteoarthritis: part III: changes in evidence following systematic cumulative update of research published through January 2009, Osteoarthr. Cartil. 18 (4) (2010) 476–499, http://dx.doi.org/10.1016/j.joca.2010.01.013. [8] G. Singh, Gastrointestinal complications of prescription and over-the-counter nonsteroidal anti-inflammatory drugs: a view from the ARAMIS database, Am. J .Ther. 7 (2) (2000) 115–121, http://dx.doi.org/10.1016/j.joca.2010.01.013. [9] E. Kon, et al., Non-surgical management of early knee osteoarthritis, Knee Surg. Sports Traumatol. Arthrosc. 20 (3) (2012) 436–449, http://dx.doi.org/10.1007/ s00167-011-1713-8. [10] T. Wright-Carpenter, et al., Treatment of muscle injuries by local administration of autologous conditioned serum: a pilot study on sportsmen with muscle strains, Int. J. Sports Med. 25 (8) (2004) 588–593, http://dx.doi.org/10.1055/s-2004-821304. [11] N. Darabos, et al., Correlation between synovial fluid and serum IL-1b levels after ACL surgery–preliminary report, Int. Orthop. (SICOT) 33 (2) (2009) 413–418, http://dx.doi.org/10.1007/s00264-008-0649-1. [12] N. Darabos, et al., Intra-articular application of autologous conditioned serum (ACS) reduces bone tunnel widening after ACL reconstructive surgery in a randomized controlled trial, Knee Surg. Sports Traumatol. Arthrosc. 19 (1) (2011) 36–46, http://dx.doi.org/10.1007/s00167-011-1458-4. [13] C. Becker, et al., Efficacy of epidural perineural injection with autologous conditioned serum for lumbar radicular compression: an investigator-initiated, prospective, double-blinded, reference-controlled study, Spine 32 (24) (2007) 1803–1808, http://dx.doi.org/10.1097/BRS.0b013e318165987d. [14] A. Barreto, et al., A new treatment for knee osteoarthritis: clinical evidence for the efficacy of Arthrokinex™ autologous conditioned serum, J. Orthop. 14 (2017) 4–9, http://dx.doi.org/10.1016/j.jor.2016.10.008.
3
Contents lists available at ScienceDirect
Cytokine journal homepage: www.elsevier.com/locate/cytokine
Short communication
A short report on the effect of decreased incubation time on the architectural profile of autologous conditioned serum (ACS) Angelique Barreto, MD Memorial Clinical Research, Oklahoma City, OK, USA
A R T I C L E I N F O
A B S T R A C T
Keywords: Autologous conditioned serum Osteoarthritis IL-1-Ra IL-1 IRAP
If present in high enough concentrations, IL-1-Ra has the potential to inhibit Interleukin-1, the chief offender that promotes the pro-inflammatory cascade causing pain, swelling and joint dysfunction associated with osteoarthritis (OA). IL-1-Ra and growth factor levels were quantified from whole blood in this retrospective chart review investigation (n = 20) using Zero and 15 min incubation times respectively. The hypothesis that this process can significantly (p < 0.0001) increase levels of IL-1-Ra was confirmed. Mean Arthrokinex™ induced IL1-Ra levels reached a concentration of 13,288 pg/mL and 12,809 pg/mL compared to 518 pg/mL at baseline, representing a 26-fold increase. Post conditioning levels of pro-inflammatories IL-1β, IL-6 and TNF α were not changed to any significant degree. The Arthrokinex™ blood conditioning process induces adequate levels of IL-1Ra to alter the IL-1-Ra: IL-1β ratio and mitigate the inflammatory cascade, while increasing growth factors PDGF and TGF respectively.
1. Introduction Traditionally, the clinical characteristics of osteoarthritis (OA) were thought to be part of the normal aging process and often classified incorrectly as degenerative joint disease. More recent studies have revealed the complex inflammatory mechanisms that cause articular cartilage destruction, sub-chondral bone thickening and synovitis. Despite these advances, the multi-factorial pathogenesis of OA, estimated to affect 27 million people in the US [1], is still poorly understood. Currently recommended pharmacologic interventions are aimed solely at symptomatic control forcing many patients to undergo surgery. Regenerative therapies such as Platelet Rich Plasma (PRP) injections are on the rise despite conflicting evidence. Given the limited effectiveness of analgesic drugs, NSAIDs and intra-articular corticosteroid injections, as well as the significant financial burden associated with treating an aging population, development of a chondroprotective drug is needed to augment joint function and reduce pain in the 67 million people OA is estimated to affect by 2030. Interleukin-1 (IL-1) is a pro-inflammatory cytokine that affects nearly every cell type [2]. Overwhelming evidence from in vitro and in vivo studies implicate IL-1β as the key mediator of the inflammatory process in OA [3]. Chondrocytes embedded within articular cartilage matrix are influenced by a variety of cytokines and growth factors that facilitate destruction and synthesis of new matrix proteins [4]. Under conditions of disease, dysregulation of this delicate balance, chiefly between IL-1β and its naturally occurring inhibitor, Interleukin-1
Receptor antagonist (IL-1-Ra), leads to matrix degradation. The Arthrokinex™ formulation has proven to consistently induce autologous conditioned serum(ACS) containing extremely high levels of the antiinflammatory cytokine, IL-1-Ra, as well as growth factors that promote cartilage regeneration [5]. Previously, a 30 min incubation time was reported as necessary to induce a 32-fold increase in IL-1-Ra from baseline. The purpose of this investigation is to determine if decreasing the incubation time has an effect on the architectural profile of ACS. It is hypothesized that decreasing the incubation time will decrease levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) and will produce significant elevations of anti-inflammatory cytokine levels (IL-1-Ra and IL-10). 2. Methods 2.1. Study design Data collection was conducted in two separate locations in Oklahoma City, OK, USA and one location in Austin, TX, USA between March 2014 and July 2016. All aspects of this retrospective chart review were reviewed and approved by IntegReview Institutional Review Board (IRB) as being considered exempt from requiring IRB approval as it met all requirements outlined in 45 CFR 46.101(b)(4), specifically (1) the research involved only the collection or study of preexisting data, documents, records, pathologic specimens or diagnostic specimens and (2) the information was recorded in such a manner that
E-mail address: [email protected]. http://dx.doi.org/10.1016/j.cyto.2017.03.019 Received 26 February 2017; Accepted 31 March 2017 1043-4666/ © 2017 Published by Elsevier Ltd.
Please cite this article as: Barreto, A., Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.03.019
Cytokine xxx (xxxx) xxx–xxx
A. Barreto
the subjects could not be identified directly or through identifiers linked to the subjects.
Table 1 Cytokine and growth factor levels of pre and post ACS conditioned serum. Serum
Arthrokinex™ Induced ACS
N = 20
No incubation time N = 10
15 min incubation time N = 10
518
13,288a
12,809a
< 3.9 < 7.8 < 15.6
< 3.9 < 7.8 < 15.6
< 3.9 < 7.8 < 15.6
< 3.1 25,892 25,226 96
< 3.1 28,163a 52,161a 94
< 3.1 32,096a 62,101a 106
2.2. Participants A total of 20 patients with symptomatic KOA met the American College of Rheumatology (ACR) inclusion criteria for analysis. According to the ACR criteria, patients with at least three of the six primary clinical features were diagnosed with KOA. These symptoms include age > 50, morning stiffness < 30 min duration, crepitus on active range of motion, tenderness of the bony margins of the joint, bony enlargement noted on examination and a lack of palpable warmth of the synovium. Exclusion criteria included: patient charts of those in generally poor health; drug dependent (chronic opioid use, alcohol, etc.); undergone surgery or treatment of the affected joint within the last 3 months; systemic disease of the musculoskeletal system; bone cancer; metastasis or tumor-like lesions in the immediate proximity to the treated joint; fracture in the last 3 months; acute bacterial infection; blood clotting disorders; continuous corticoid or NSAID therapy due to other diseases. Informed consent was obtained from each participant and all work was performed in accordance with The Code of Ethics of the World Medical Association (Declaration of Helsinki, 1975 revised 2008).
IL-1-Ra (pg/ mL) IL-1β (pg/mL) IL-10 (pg/mL) TNF-α (pg/ mL) IL-6 (pg/mL) TGF (pg/mL) PDGF (pg/mL) IGF-1 (ng/mL)
a Statistically significant (p value < 0.0001) elevation when compared to pre conditioned serum.
2.5. Statistical analysis SPSS 12.0 for Windows (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The non-parametric Wilcoxon Signed Rank test was performed to analyze the statistical difference between baseline and post-processing cytokine levels. All results shown are the mean ± SEM of two or more experiments.
2.3. Processing of whole blood Using aseptic techniques, 60 mL venous whole blood of twenty (20) participants was harvested into a sterile 60 mL syringe containing 3 mL of anticoagulant citrate dextrose (ACD) solution and centrifuged (3200 rpm, 15 min). The resultant Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP) were extracted and the remaining layer containing erythrocytes, was discarded. Both the PRP and PPP were transferred to a specialized, closed-system, centrifuge tube containing medical grade concentrator beads and mixed. Ten samples were incubated for 15 min and 10 samples were incubated for zero minutes. After the reduced incubation period, centrifuge filtration (2000 rpm, 3 1/2 min) through a sterile 0.45 µm filter was completed and the resulting sterile filtrate obtained. The ACS could be used immediately for intra-articular injection or stored at −20 degrees Celsius for future use.
3. Results and discussion The purpose of this retrospective analysis was to test the hypothesis that pro-inflammatory cytokines would decrease, extremely high levels of IL-1-Ra would be maintained and growth factors (IGF-1, TGF and PDGF) would increase after reducing the incubation time of the formulation. This hypothesis was proven correct. All pro-inflammatory cytokine levels read below the minimum value at each incubation time (Table 1). At the previous incubation time of 30 min, pro-inflammatory cytokine levels of post-Arthrokinex™ were higher than pre-conditioned serum. IL-1-Ra levels at these shorter incubation times were similar to serum conditioned at the original 30 min [5]. Additionally, PDGF and TGF levels were significantly increased following the Arthrokinex™ formulation at each incubation time. The presence of these growth factors contained in ACS supports the hypothesis that IA injections of ACS may reverse the disease process associated with OA as growth factors have been shown to stimulate chondrocyte proliferation and augment articular cartilage metabolism. At increased concentrations, IL-1-Ra has the potential to limit the destructive inflammatory intra-articular actions of IL-1β. IL-1-Ra concentrations must exceed IL-1β levels by 10–100 fold to competitively inhibit the interaction of IL-1β with its receptors. On average, knees affected with OA will contain approximately 34 pg of IL-1β [6]. This novel method provided de novo levels of IL-1-Ra that exceeded the 100:1 threshold in each of the 10 serum samples incubated at 15 min and each of the 10 serum samples incubated for zero minutes. Given the significantly increased levels of IL-1-Ra incubated for 15 min (12,809 pg/mL), the significantly increased levels of IL-1-Ra incubated at zero minutes (13,288 pg/mL), and the ratio of the Arthrokinex™ induced IL-1-Ra to the pro-inflammatory cytokine (IL-1β), it is reasonable to conclude that autologous conditioned serum(at each incubation time) can consistently antagonize IL-1β receptor binding. While at first glance this approach may appear similar to other commercially available products, this process has several important modifications. Initially, this novel formulation was designed to incubate for 24 h. However, after several attempts, it was discovered that the same levels of IL-1-Ra could be induced after the PRP/PPP mixture was in contact with the beads for only 30 min. Currently, it is demonstrated that 15 min and even zero minutes of incubation time are sufficient to
2.4. Biomarker assays The primary outcome of measuring IL-1-Ra (pre- and post-conditioning) was achieved by using the highly sensitive, commercially available quantitative sandwich enzyme-linked immunoassay technique (R & D Systems, Quantikine ELISA; Minneapolis, MN, USA). This kit, when run in accordance with standard Quantikine protocols, is extremely sensitive (minimum detectable dose ranged from 2.2 to 18.3 pg/mL), specific (no significant cross-reactivity or interference was observed), precise (intra- and inter-assay CVs were 3.7% and 6.7%) and linear (all diluted samples fell within the dynamic range of the assay). Resulting concentrations were calculated by subtracting the average zero standard optical density and log transforming IL-1-Ra concentrations versus the log of the optic density on a linear scale, and the best fit line determined by regression analysis. IL-1-Ra concentrations were only accepted if the standard curve correlation coefficient (r) reached 0.99 and the CV of each sample was under 20%. Additionally, serum levels (pre- and post-conditioning) of proinflammatory (TNF α, IL-1β, IL-6) cytokines, anti-inflammatory cytokine (IL-10) and growth factors (insulin like growth factor 1, platelet derived growth factor and transforming growth factor) were also measured using ELISA. All kits reported comparable sensitivity, specificity, precision and linearity as described above and were run in accordance with standard Quantikine protocols. 2
Cytokine xxx (xxxx) xxx–xxx
A. Barreto
clinical trials are needed to compare the effects of Arthrokinex™ (ACS) to other currently accepted therapeutic options. Meanwhile, this shorter incubation time offers an alternative, on-site, point of service treatment option for a population with an increasing burden of OA.
produce high quality ACS. Moreover the shorter incubation time decreased the pro-inflammatory cytokine profile and maintained IL-1Ra levels. Clinically this would translate into continued pain relief and less chance of a post IA injection flare. Additionally, Arthrokinex™ does not introduce any additional chemicals and utilizes a closed loop system to avoid contamination. The lack of effective treatment options for OA continues to present a challenge for patients and providers. Acetaminophen is the first line oral option available to treat mild to moderate knee OA [7] even though evidence suggests that NSAIDs have superior analgesic effects. NSAIDs are recommended for moderate to severe OA but should be used judiciously due to the well-known gastro-intestinal, renal and cardiotoxic effects [8] including stroke and myocardial infarction. The Osteoarthritis Society International (OARSI) [7] also recommends the administration of short term IA corticosteroid injections; however, serious side effects of steroid [9] injections should be considered. The chronic nature of OA and availability of short term treatment options highlights the need for a chondroprotective agent that can modify the disease process at the molecular level. Joint injections of serum rich in IL-1-Ra and growth factors, induced by this blood conditioning process, has the potential to shift the intra-articular conditions to a more favorable cytokine profile. Furthermore, this process is relatively inexpensive with an expected favorable safety profile given the biotherapeutic approach. Several clinical trials highlight other potential applications of the Arthrokinex™ process beyond the treatment of OA. Clinical improvements have been reported in muscle and ligament injury [10] and spinal degenerative disk disease [13]. Darabos et al. [11,12] demonstrated increased levels of IL-1β in the synovial fluid of 19 out of 20 knees following ACL reconstructive surgery, and resultant reduction in post surgery complications following administration of IL-1-Ra IA injections. Mitigating post-surgical inflammation could be accomplished by administering Arthrokinex™ injections. Given the overwhelming data describing many different clinical approaches of IL-1Ra, the potential of autologous conditioned serum is broad. It is important that a notable limitation due to the short half-life of the serum exists, and is overcome in large part by the administration of closely spaced IA injections of stored serum in the Arthrokinex™ clinical protocol. It was recently demonstrated that intra-articular injections of Arthrokinex™ (ACS) provided significant clinical benefits in a clinical trial involving 100 patients with symptomatic KOA [14]. Additional
References [1] C.G. Helmick, et al., Estimates of the prevalence of arthritis and other rheumatic conditions in the US, Arthritis Rheum. 58 (1) (2008) 15–25, http://dx.doi.org/10. 1002/art.23177. [2] C. Dinarello, Interleukin-1, Cytokine Growth Factor Rev. 8 (4) (1997) 253–265, http://dx.doi.org/10.1016/S1359-6101(97)00023-3. [3] M. Goldring, Osteoarthritis and cartilage: the role of cytokines, Curr. Rheumatol. Rep. 2 (6) (2000) 459–465, http://dx.doi.org/10.1007/s11926-000-0021-y. [4] L.C. Dijkgraaf, L.G. de Bont, G. Boering, R.S. Liem, Normal cartilage structure, biochemistry, and metabolism: a review of the literature, J. Oral Maxillofac. Surg. 53 (8) (1995) 924–929, http://dx.doi.org/10.1016/0278-2391(95)90283-X. [5] A. Barreto, et al., A method to induce Interleukin 1 receptor antagonist from autologous whole blood, Cytokine 81 (2016) 137–141, http://dx.doi.org/10.1016/ j.cyto.2016.03.008. [6] G.S. Firestein, J.M. Alvaro-Gracia, R. Maki, Quantitative analysis of cytokine gene expression in rheumatoid arthritis, J. Immunol. 144 (9) (1990) 3347–3353, http:// dx.doi.org/10.1008/s13567-028. [7] W. Zhang, et al., OARSI recommendations for the management of hip and knee osteoarthritis: part III: changes in evidence following systematic cumulative update of research published through January 2009, Osteoarthr. Cartil. 18 (4) (2010) 476–499, http://dx.doi.org/10.1016/j.joca.2010.01.013. [8] G. Singh, Gastrointestinal complications of prescription and over-the-counter nonsteroidal anti-inflammatory drugs: a view from the ARAMIS database, Am. J .Ther. 7 (2) (2000) 115–121, http://dx.doi.org/10.1016/j.joca.2010.01.013. [9] E. Kon, et al., Non-surgical management of early knee osteoarthritis, Knee Surg. Sports Traumatol. Arthrosc. 20 (3) (2012) 436–449, http://dx.doi.org/10.1007/ s00167-011-1713-8. [10] T. Wright-Carpenter, et al., Treatment of muscle injuries by local administration of autologous conditioned serum: a pilot study on sportsmen with muscle strains, Int. J. Sports Med. 25 (8) (2004) 588–593, http://dx.doi.org/10.1055/s-2004-821304. [11] N. Darabos, et al., Correlation between synovial fluid and serum IL-1b levels after ACL surgery–preliminary report, Int. Orthop. (SICOT) 33 (2) (2009) 413–418, http://dx.doi.org/10.1007/s00264-008-0649-1. [12] N. Darabos, et al., Intra-articular application of autologous conditioned serum (ACS) reduces bone tunnel widening after ACL reconstructive surgery in a randomized controlled trial, Knee Surg. Sports Traumatol. Arthrosc. 19 (1) (2011) 36–46, http://dx.doi.org/10.1007/s00167-011-1458-4. [13] C. Becker, et al., Efficacy of epidural perineural injection with autologous conditioned serum for lumbar radicular compression: an investigator-initiated, prospective, double-blinded, reference-controlled study, Spine 32 (24) (2007) 1803–1808, http://dx.doi.org/10.1097/BRS.0b013e318165987d. [14] A. Barreto, et al., A new treatment for knee osteoarthritis: clinical evidence for the efficacy of Arthrokinex™ autologous conditioned serum, J. Orthop. 14 (2017) 4–9, http://dx.doi.org/10.1016/j.jor.2016.10.008.
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