TSG-6

TSG - 6

Is TSG-6 the most important biomolecule in orthopedic molecular immunobiologics?

Austin Yeargan III MD

Background:

Bone marrow concentrates for knee arthritis have received much attention over the last decade as a viable treatment for osteoarthritic knee pain and disability.  In spite of the availability of these bone marrow concentrates, there has yet to be a gold standard for treatment suggested.  We first introduced our signaling cell procedure to orthopedic surgery in 2006.  The procedure has undergone multiple iterations since that time.  In this article, we introduce our seventh-generation technique for signaling cell treatment in the setting of osteoarthritis of the knee and discuss the role of tumor necrosis factor stimulated gene six protein in our signaling cell product. 

We identified TSG-6 as an important chondroprotective biomolecule that is critical for the assembly and maintenance of the cartilage extracellular matrix.  TSG-6 is an inflammation-induced protein that is produced at pathological sites, like synovial joints during arthritic degeneration.  TSG-6 protects against joint damage through anti-plasmin activity and de-activation of serine protease during inflammation.   TSG6 also limits neutrophil migration and has an anabolic, immunomodulatory effect in synovial joints.[1]

Modern signaling cell techniques are unable to capture or concentrate TSG-6 due to its small molecular size, that is coincident undesirable with pro-inflammatory molecules.  These molecules are on the order of 30 kD in comparison with anti-inflammatory molecules that typically are greater than 600 kD in size.  Hyaluronic acid typically has a molecular weight of 3000-4000 kD and is bound by one of the domains of TSG-6, causing it’s biological activation.

We combine our platelet poor plasma and a commercially prepared hyaluronic acid to formulate a growth factor concentrate that is activated as a scaffold for signaling cells we harvest from autologous bone marrow aspirate taken at the anterior gluteal pillar.  We believe that by concentrating TSG-6 and including it as a component of our signaling cell transplant procedures, a biologically superior injectate is able to be assembled.  In addition, we harvest autologous clotting proteins to lock the product in the subchondral bone after micro core of the stiff, subchondral bone using a commercially available device. 

Introduction:

Tumor necrosis factor (TNF) stimulated gene 6, or TSG6, the only biochemical to carry a “chondroprotective” designation, is often overlooked and is almost universally lost in translation from bench science to clinical application of orthopedic immunobiologics, even with 5th and 6th generation techniques for signaling cell product concentration.  Here we describe the mechanism of action of TSG6 and propose how that translates to the treatment of joint arthritis and subchondral remodeling with our algorithm for nanoplasty† and mechanical axis deviation (NAMAD®) previously described by us as a technique to manage unicompartmental gonarthrosis with MicroCore†.

Hyaluronan is the activator of TSG-6.  We explain the role of activated TSG-6 in the setting of arthritis.  Because the canonical and non-canonical pathways of both inflammatory (Rheumatoid type arthritis or RA) and load dependent arthritis (osteoarthritis or OA) have a common signaling pathway tree, the biomolecule is critical in the setting of both processes to protect the articular surface regardless of the arthritic condition.  We have developed a simple technique for capturing and concentrating TSG6 in the setting of orthopedic immunobiologics for arthritis and arthritic joint pain using an FDA approved drug (HA) while maintaining adherence to strict standards of ‘minimal manipulation’ of harvested and concentrated bone marrow put forth by the FDA concerning the regulatory considerations for human cells, tissues, and cellular and tissue-based products: Minimal Manipulation and Homologous Use.  In other words, we strictly adhere to Title 21 of the Code of Federal Regulations (CFR) Part 1271, specifically the 21 CFR 1271.10(a)(1) criterion of minimal manipulation and the 21 CFR 1271.10(a)(2) criterion of homologous use.[2] 

Our process is FDA cleared for safety.  Application of cell processors in the setting of signaling cell transplantation for arthritis constitutes off-label use.  This fact is made perfectly clear to any patient who is considering or who is offered our treatment algorithm, which has strict criteria of its own.  While the science is significant, verification of clinical translation to improved patient care have not been scientifically investigated or proven by anyone other than us, to our knowledge.  We are currently compiling our data to determine whether our unique approach to arthritis actually halts the progression of organic joint disease or simply relieves the symptoms through previously described nanomolecular, immunomodulatory, anti-inflammatory mechanisms.

TSG6 is a 35000 Da, or 35 kDa protein composed of contiguous Link and Complement 1r/s, Sea Urchin epidermal growth factor (Uegf) and BMP (CUB) modules. [3] [4]  CUB modules are ubiquitous, multifunctional extracellular domains found in 16 functionally diverse proteins.  Most are involved in developmental pathways.  Link modules are found in many proteins that interact with the ubiquitious glycosaminoglycan (GAG) Hyaluronic acid (HA), a vital component of the extracellular cartilage matrix that chondrocytes elaborate, reside in and migrate through.[5]  The HA binding site of Link-TSG6 has been mapped by nuclear medicine spectroscopy and site-directed mutagenesis, identifying five key residues contributing to HA binding.[6] [7] [8] [9]  It is now clear that BMP-1 domains act in dorsal-ventral patterning via activation of transforming growth factor beta (TGFbeta)-like proteins BMP2, BMP4 and play a major role in patterning of extracellular and intracellular matrix formation.[10]  In vertebrates, the BMP1/TLD-like metalloproteinases play key roles in regulating formation of the extracellular matrix (ECM) via biosynthetic processing of various precursor proteins into mature functional enzymes, structural proteins, and proteins involved in initiating mineralization of the ECM of hard tissues.[11] 

 

Extracellular matrix (ECM) remodeling is the key feature of TSG6 production that draws our attention in the clinical setting of orthopedic immunobiologics.  TSG6 interacts with ECM GAG and HA as well as two other ECM components, Chondroitan-4-sulfate and aggrecan.[12]  TSG6 also binds the serine protease inhibitor, inter-alpha-inhibitor (IaI), which leads to crosslinking of hyaluronan chains and increased anti-plasmin activity, limiting matrix destruction.  TSG-6 is a novel target that may offer structural matrix stability when combined with axial load management strategies that reverse gravity induced arthritic remodeling due to chronic inflammatory signaling.  The clinical efficacy of hyaluronic acid is likely attributable to the presence and concentration of TSG-6 characterizing arthritis.

TSG-6 is presents in bone marrow aspirate and concentrates in the platelet poor plasma fraction of the isopycnic product.  Even more advanced signaling cell transplant procedures that eliminate pro-inflammatory cytokines and preserve growth factors and healing response molecules through nanofiltration fail to conserve TSG-6 due to its molecular size exclusion.  Commercial nanofilters have pore sizes that are approximately 50-60 kD and were designed to capture Alpha-2 macroglobulin, once described as the master molecule of inflammation.[13]  We believe TSG-6 could be a more important biomolecule that should be preserved and concentrated in every case of signaling cell transplant for arthritis.

We designed and developed a specific signaling cell harvesting and processing protocol to capture and concentrate TSG-6 proven using enzyme linked immunosorbent assay techniques previously described.  We describe the application of our protocol to human orthopedic science and translation to a clinically viable algorithm for signaling cell transplantation in the setting of symptomatic human arthritis of the knee.

Tumor necrosis factor stimulated gene 6, or TSG6 is the only biochemical to carry a “chondroprotective” designation and may be the most important molecule in cartilage and joint metabolism when considered at the quantum, or nanomolecular, level.  The immunomodulatory mechanisms of TSG-6 serve to protect articular hyaline cartilage from destruction and amplify anabolic cell signaling that favorably affect sapien symptoms arising from arthritic joint disease.  As the name implies, TSG6 is indeed stimulated by the presence of tumor necrosis factor and interleukin-1 in joint fluid that is secreted by type A synoviocytes after mechanochemical co-activation.[14]

Arthritis is a naturally progressive process that comes from the body’s biological cellular response to gravitational overload that occurs because of attempts to stabilize the physical machine against the pull of gravity.  This constant 9.8 m/s2 instantaneous force is countered by musculotendinous force vectors experienced with bipedal human locomotion that contribute to tissue overload.  These gross force vectors that follow Newtonian physics are translated biomechanically to biochemical signals that dictate physical roles at the nanomolecular level, where particle physical laws predict outcome.  Thanks to technology like single cell proteomics, flow cytometry and ELISA, we have been able to dissect the immunomodulatory signaling pathways that underlie arthritic diseases in the human under both primary and secondary inflammatory conditions.  Understanding these signaling pathway and their overlap with the basic metabolic requirements of other cells and tissues has expanded therapeutic opportunity in biologics in every field in medicine. 

Mechanotransduction and the genesis of TSG-6 secretion

In the setting of matrix destruction, like that seen in arthritic conditions, the secretion of tumor necrosis factor (TNF-a) and interleukin-1 (IL-1B) by type A synoviocytes (monocytes) prompts TSG production by chondrocytes, type B synoviocytes (fibroblasts) and vascular smooth muscle cells.  TSG6 is rapidly upregulated by the presence of pro-inflammatory cytokines like IL-6 and IL-8 and serves as a spatiotemporal buffer to matrix metalloproteinase destruction during the first phase of inflammation. Overexpression of TSG6 causes an increase in progenitor cell proliferation, which confirms its potential role in the clinical management of arthritis where conversion to anabolic metabolism is favorable. [15]

TSG6 maps to human chromosome 2q23.3.[16] [17]  Synthesis is tightly regulated in a wide variety of cell types.  Perhaps more important in the context of joint disease, growth factors like TGFB, EGF, FGF upregulate TSG6 in some cell types like chondrocytes.[18]  Interestingly, TSG6 does not have osteogenic properties. 

TSG-6 is a member of the Link module superfamily and binds hyaluronan, and other glycosaminoglycans a vital part of the extracellular matrix by the link module.  TSG-6 forms covalent and non-covalent complexes with inter-alpha-inhibitor, which is a serine protease inhibitor (SERPIN), potentiating its alpha-2 antiplasmin activity.  Plasmin is an important enzyme that participates in fibrinolysis and protein catabolism.[19] 

TSG6 is not constitutively secreted.  Like endocannabinoids, it is secreted on demand by many of the cells involved in the immunomodulatory, anti-inflammatory cascade, that naturally follows the pro-inflammatory cascade.  For our purposes, we are interested in the active secretion of TSG-6 by both synovial inflammatory cells (monocytes) and mesenchymal stem cells (MSCs).  TSG6 is produced in the context of inflammatory mediators and is not found in healthy adult tissues.  Interestingly, TSG6 expression is induced by PGE2, the only prostaglandin with an anabolic designation.[20] 

TSG6 mediates the function of TDSCs to improve the structure and the attachment strength of the healing tendon-bone interface.  TSG6 can be found in the synovial joint fluid, interstitial tissue fluid and even in the sera of patients with arthritis, pointing to a potential point-of-care marker for arthritis.[21]

TS6 is a small biomolecule with a molecular weight of approximately 35 kD.  It is the smallest of the anti-inflammatory biochemicals, and perhaps one of the most important.  Alpha-2-macroglobulin, often called the ‘master regulator’ has a molecular weight of 800 kDa and is typically snagged and concentrated using commercially available nanopore filters with a pore size of 65 kDa when advanced cell concentrating techniques are applied clinically.  With a molecular weight of 30 kDa, the native TSG-6 is lost during nanofiltration due to an inability to be caputured based on size exclusion principles.

 

Since the anti-inflammatory cascade is part of the inflammatory cascade, it cannot be manipulated in vivo for therapy without first concentrating higher molecular weight anti-inflamatory proteins in vitro before returning them as an immunobiologic product as described in this treatment.  Without TSG-6, the full anti-inflammatory complement is absent and may compromise the repair/regeneration stage of inflammation, which could affect the long-term remodeling third phase that concludes the process where extracellular matrix elaboration is critical.  Modulation of the repair/regeneration and remodeling stages may lead to less fibroblastic scarring with more desirable tissue and better biomechanical properties.  TSG6 appears in vascular smooth muscle cells following musculosketal injury and mechanical strain.

The problem of TSG-6 exclusion in this setting is obvious.  Our efforts to design and develop a nanofilters that would retain TSG-6 and was met without success initially.  Once we understood the importance of the biomolecule, our attention was focused on how to capture and concentrate it.

My ’secret’ remedy has been to mix the marrow plasma with hyaluronic acid BEFORE I passage the marrow plasma across the filter.  The HA binds to and activates TSG-6, which restricts a serine protease. 

TSG-6 is the only biochemical that has a ‘chondroprotective’ designation.  It’s interesting that no one has commented on the mechanism of action of HA being the activator of TSG-6, although that’s pretty easy to run down. Very easy in my experience.  There seems to be a disconnect between the scientists and orthopedic surgeons in that regard. Maybe I’m wrong. 

We have proven that the step does concentrate TSG-6.  I have done ELISA on the samples at a lab here that is run by the University of NC at Wilmington’s ’SeaTox®’ lab.  

I do this on everyone now.  I also go into the bone on the concave side of the joint, which decompresses the bone (like AVN, which it is formally), I inject the product and use autologous thrombin that I collect with the Arthrex® Throminator, which I think is a big step forward in this process.  It’s more expensive, but I think that it’s better clinically than using cow thrombin if you don’t have to.  

How does TSG-6 work?

TSG-6 is upregulated in many cell types in response to pro-inflammatory mediators and growth factors.[22]  The protein has specific application in joint disease due to it’s chondroprotective effects in models of arthritis where extracellular matrix remodeling appears to be it’s primary responsibility.  TSG-6 is part of a negative feedback loop that is capable of both matrix stabilization and immunomodulatory downregulation.

How do we set a molecular trap for TSG6?

TSG-6 is secreted by synovial type A cells in response to elevated levels of TNF-alpha and interleukin 1-Beta concentration ratios.  Elevated levels of TNFå and IL-1B secretion come at the expense of physical matrix degradation and recently enhanced proteome sequencing has revealed an interesting phasic response based on load application in both weight bearing and non-weight bearing joints that are susceptible to inflammatory conditions as the result of a non-concentric articular loading condition and mis-directed musculoskeletal force vectors. 

Why is it important?

Earlier pain relief

Better autologous scaffold construction. 

What we do:

TSG-6 is perhaps the most important molecule in cartilage repair metabolism and is the only biochemical molecule with the designation “chondroprotective”.  TSG-6 shares a similar molecular weight to all of the pro-inflammatory proteins in the 25-30 kD range.  TSG-6 is not found in articular fluid until arthritis has been initiated by an inflammatory process, as determined by the presence of TNF-a and IL-1.  Our lab has performed quantitative assays for TSG-6 in bone marrow aspirate, platelet poor plasma, growth factor concentrate and bone marrow concentrate.  These assays have demonstrated the presence of the protein in bone marrow and platelet poor plasma as well as the growth factor concentrate.  We have demonstrated the ability to concentrate TSG-6 using a very simple, FDA-approved, third party payer reimbursed procedure.  We believe this next generation modification should be performed with every cell transplant procedure that is done.  Our early clinical data is encouraging and the technique may represent part of a best clinical practice model for an evolving gold standard in cell based therapy (CBT) management.  Prior to using the next generation techniques we now employ, patients reported improvement of symptoms within three weeks.  With next-gen TSG-6 capture, patients have unequivocally reported relief by the next day, if not the afternoon of their procedure.  We believe there is a major role for an unmet need where arthritis is concerned.  Autologous biologics are based in science and on immunologic signaling pathways.   We describe a method that is safe, cost effective, scientifically based and clinically proven, at a cost far less than the biosimilars demand. 

Biomechanical interventions with an intent to reverse subchondral bone stiffness are critical.  i a protocol driven program including physical therapy. 

We have developed a novel technique to capture TSG-6 in spite the fact that TSG-6, unlike other anti-inflammatory cytokines, matches the molecular weight of the pro-inflammatory molecules.   TSG-6 does not appear in normal synovial fluid.  As its name implies it is not released until tumor necrosis factor alpha and interleukin-one (IL-1)  activate fibroblast-like synovial lining cells (FLS). 

Established programs should have written, consistent protocols including the use of a few therapists. .

Current commercially available bone marrow processors intended to capture the nucleated signaling cell population does not retain proteins with MWCO of less than 65000, or 6500 kDa.

With the more specific filtration we describe that captures and concentrates TSG6, a better Biologics product for delivery to a orient in the point-of-care setting

Specifically, TSG6 and transforming growth factor Beta, both conclusively demonstrated to be critical components of cartilage metabolism.  Progenitor cells will not specialize into chondrocytes in the absence of TGF-B. 

Incorporating a polyethersulfone filter with MWCO of 15-20 kD would allow the capture of a characterizable protein component that does include concentrated TSG6 and TGFB.

I believe that custom engineering an autologous scaffold from the sample is essential in delivering the best possible Orthobiologics product.  Fibrin forms a basis for cells to anchor via integrins and cadherins.  Conglomeration of cells sets up a relatively hypoxic environment that encourages cell adhesion upon injection.  The injectate may be more effective if activated by an appropriate CaCl (or CaGluconate) and thrombin concentration.

Mechanotransuction comes from adherence of cells to each other and to the matrix molecules they elaborate.  Protein cadherins and integrins account for cell-to-cell and cell-to-matrix interactions that are load responsive.  Activation of mechanoreceptors leads to intracellular signaling and spatiotemporally based immunomodulation of living tissues.  Coupled with subchondral mechanotransduction is the osteoclastic resorption front that leads to osteoblast migration and osteocyte and matrix formation in the challenged subchondral bone.  Additional supportive bone is laid down in a support column corresponding to the mechanical axis and bordered by the physeal scar, where mechnoreceptors are concentrated on loaded bone and cartilage cells.  This feature is easily seen on terracon MR image sequences.   Otherwise the concave, elastic subchondral bone is remodeled and takes on the material characteristics of cortical bone, which is much more stiff and unable to absorb the impact shock from the convexity.  As additional bone is remodeled, intraosseous pressures become elevated and oppose new vascular flow and ingrowth, compounding the problem as subchondral bone becomes relatively avascular.

 

[1] Qu F, Guilak F, Mauck RL. Cell migration: implications for repair and regeneration in joint disease. Nat Rev Rheumatol. 2019;15(3):167–179. doi:10.1038/s41584-018-0151-0

 

[2] https://www.fda.gov/vaccines-blood-biologics/biologics-guidances/tissue-guidances

 

[3] Lee HX, Mendes FA, Plouhinec JL, De Robertis EM. Enzymatic regulation of pattern: BMP4 binds CUB domains of Tolloids and inhibits proteinase activity. Genes Dev. 2009;23(21):2551–2562. doi:10.1101/gad.1839309

 

[4] Bork P, Beckmann G.  The CUB Domain. A widespread module in developmentally regulated proteins.  J Mol Biol 1993 May 20:231(2): 539-45.

 

[5] Tammi MI, Day AJ, Turley EA.  Hyaluronan and Homeostasis: a balancing act. J Biol Chem 2002 Feb 15; 277(7): 4581-4. Epub 2001 Nov 20.

 

[6] Mahoney DJ, Blundell CD, Day AJ.  Mapping the hyaluronan-binding site ont eh link module from human tumor necrosis factor-stimulated gene-6 by site-directed mutagenesis.  J Biol Chem 2001 Jun 22;276(25): 22764-71. Epub 2001 Apr 3.

 

[7] Getting SJ, Mahoney DJ, Cao T, Rugg MS, Fris E, Milner CM, Perretti M, Day AJ.  The link module from human TSG-6 inhibits neutrophil migration in a hyaluronan-and inter-slpha-inhibitor-independent manner. J Biol Chem 2002 Dec 27; 277(52): 1068-75.

 

[8] Kahman  JD, O’Brien R, Werner JM, Heinegard D, Ladbury JE, Campbell ID, Day AJ.  Localization and characterization of the hyaluronan-bindign site on the link module from human TSG-6.  Structure 2000 July 15;8(7): 763-4.

 

[9] Fowlkes JL, Serra DM. Charaterization of Glcosaminoglycan-binding domains present in Insulin-like Growth Factor-binding Protein 3.  J Biol Chem. 1996 Jun 21;271(25): 14676-14679.

 

[10] Hopkins D, Keles S, Greenspan DS. The bone morphogenic protein 1/Tolloid-like metalloproteinases. Matrix Biol 2007 Sep;26(7): 508-23. Epub 2007 May 18.

 

[11] Ge G, Greenspan DS. Developmental roles of the BMP1/TLD metalloproteinases. Birth Defects Res C Embryo Today 2006 Mar;78(1): 47-68.

 

[12] Parkar AA, Day AJ. Overlapping sites on the Link module of human TSG-6 mediate binding to hyaluronan and chondroitin-4-sulphate.  FEBS Lett 1997 Jun 30;410(2-3): 413-7.

 

[13] Wang S, Wei X, Zhou J, et al. Identification of α2-macroglobulin as a master inhibitor of cartilage-degrading factors that attenuates the progression of posttraumatic osteoarthritis. Arthritis Rheumatol. 2014;66(7):1843–1853. doi:10.1002/art.38576

 

[14] Lowin T, Straub RH. Integrins and their ligands in rheumatoid arthritis. Arthritis Res Ther. 2011;13(5):244. doi:10.1186/ar3464

 

[15] Lee RT, Yamamoto C, Feng Y, Potter-Perigo S, Briggs WH, Landschulz KT, Turi TG, Thompson JF, Libby P, Wright TN.  Mechanical strain induces specific changes in the synthesis and organization of proteoglycans by vascular smooth muscle cells.  J Biol Chem 2001 Apr 27; 276(17) 13847-51. 

 

[16] Lee TH, Klampfer L, Shows TB, Vilcek J.  Transcriptional regulation of TSG6, a TNF and IL-1 inducible primary response gene coding for a secreted hyaluronan binding protein.  J Biol Chem 1993 Mar 25; 268(9)L 6154-60.

 

[17] Nentwich HA, Mustafa Z, Rugg MS, Marsden BD, Cordell MR, Mahoney DJ, Jenkins SC, Dowling B, Fries E, Milner CM, Loughlin J, Day AJ.  A novel allelic variant of the human TSG-6 gene encoding an amino acid difference in the CUB module. Chromosomal localization, frequency analysis, modeling and expression.  J Biol Chem 2002 May 3; 277(18): 15354-62. Epub 2002 Feb 19.

 

[18] Liu S, Cao C, Zhang Y, Liu G, Rn W, Ye Y, Sun T.  PI3K /Akt inhibitor partly decreases TNF-a-induced activation of fibroblast-like synoviocytes in osteoarthrtitis.  J Orthop Surg Res 2019 Dec 11;14(1): 425.

 

[19] Martin J, Midgley A, Meran S, Woods E, Bowen T, Phillips AO, Steadman R.  Tumor necrosis factor-stimulated Gene 6 (TSG6) heavy chain 5 facilitate transforming growth factor B1-dependent fibroblast to myofibroblast differentiation.  J Biol Chem 2016 May 11; 115(291): 13789-13801.

 

[20] Bartosh TJ, Ylöstalo JH, Bazhanov N, Kuhlman J, Prockop DJ. Dynamic compaction of human mesenchymal stem/precursor cells into spheres self-activates caspase-dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1). Stem Cells. 2013;31(11):2443–2456. doi:10.1002/stem.1499

 

[21] Bayliss et al. 2001

[22] CM Milner, AJ Day.  TSG-6:  a multifunctional protein associated with inflammation.  Journal of Cell Science 2003 116:  1863-1873.

Author
S. Austin Yeargan III, MD Orthopedic Surgeon Molecular Immunobiologist

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