Melissa A Hausburg1, Elizabeth D Frederick2, Patrick McNair3, John Schwappach4, Kaysie L Banton4, Michael Roshon5, Mark J Lieser6, David L Acuna7, Rahul R Banerjee8, David Bar-Or9. 1. Trauma Research Dept of Swedish Medical Center, Englewood, CO; St. Anthony Hospital, Lakewood, CO; The Medical Center of Plano, TX; Penrose Hospital, Colorado Springs, CO; Research Medical Center, Kansas City, MO; Wesley Medical Center, Wichita, KS, USA. 2. Trauma Research Department, Swedish Medical Center, Englewood, CO, and Ampio Pharmaceuticals, Inc., Englewood, Colorado, USA. 3. Trauma Research Department, St. Anthony Hospital, Lakewood, Colorado, USA. 4. Trauma Research Department, Swedish Medical Center, Englewood, Colorado, USA. 5. Trauma Research Department, Penrose Hospital, Colorado Springs, Colorado, USA. 6. Trauma Research Department, Research Medical Center, Kansas City, Missouri, USA. 7. Trauma Research Department, Wesley Medical Center, Wichita, Kansas, USA. 8. Trauma Research Department, The Medical Center of Plano, Texas, USA. 9. Trauma Research Depts. of Swedish Medical Center, Englewood, CO, St. Anthony Hospital, Lakewood, CO, The Medical Center of Plano, TX, Penrose Hospital, Colorado Springs, CO, Research Medical Center, Kansas City, MO, Wesley Medical Center, Wichita, KS; Ampio Pharmaceuticals, Inc., Englewood, CO; Dept. Molecular Biology, Rocky Vista University, Parker, CO, USA.
Abstract
OBJECTIVES: Traumatic joint injury induces chondrocyte dysfunction and progressive breakdown of articular cartilage, leading to post-traumatic osteoarthritis (PTOA). In this condition, dysfunctional fibroblast-like chondrocytes (FLCs) no longer express proteins required for cartilage maintenance, such as SOX9 and collagen-type II (COL2). Interleukin-6 (IL-6) has been demonstrated to downregulate expression of these two critical proteins in chondrocytes, and increased IL-6 levels have been measured in patients with PTOA. The <5kDa fraction of human serum albumin (LMWF5A) has been suggested to modulate this pathway, as decreased levels of IL-6 are secreted by immunostimulated LMWF5A-treated macrophages. Our objective was to determine whether LMWF5A induces an in vitro model of FLCs to redifferentiate into functional chondrocytes. METHODS: SOX9 and COL2 were monitored via western blot, and COL2 was detected with immunofluorescence. Aggrecan and IL-6 were quantified by ELISA. Glycosaminoglycan (GAG) levels were quantified with alcian blue. RESULTS: We found that LMWF5A significantly increases the principal cartilage transcription factor SOX9 and the SOX9 target protein COL2 in monolayer-cultured FLCs. Multiple LMWF5A treatments of 3-D pellet FLC cultures over 2wks resulted in a significant decrease in IL-6 and significant increases in the major players of articular cartilage mechanics, aggrecan and highly-sulfated GAGs. CONCLUSIONS: These data support the hypothesis and clinical outcomes of two phase III clinical trials that LMWF5A-treatment induces chondrogenesis and supports functional cartilage. We propose that LMWF5A could maintain articular cartilage integrity in all joints following traumatic injury.
OBJECTIVES:Traumatic joint injury induces chondrocyte dysfunction and progressive breakdown of articular cartilage, leading to post-traumatic osteoarthritis (PTOA). In this condition, dysfunctional fibroblast-like chondrocytes (FLCs) no longer express proteins required for cartilage maintenance, such as SOX9 and collagen-type II (COL2). Interleukin-6 (IL-6) has been demonstrated to downregulate expression of these two critical proteins in chondrocytes, and increased IL-6 levels have been measured in patients with PTOA. The <5kDa fraction of humanserum albumin (LMWF5A) has been suggested to modulate this pathway, as decreased levels of IL-6 are secreted by immunostimulated LMWF5A-treated macrophages. Our objective was to determine whether LMWF5A induces an in vitro model of FLCs to redifferentiate into functional chondrocytes. METHODS:SOX9 and COL2 were monitored via western blot, and COL2 was detected with immunofluorescence. Aggrecan and IL-6 were quantified by ELISA. Glycosaminoglycan (GAG) levels were quantified with alcian blue. RESULTS: We found that LMWF5A significantly increases the principal cartilage transcription factor SOX9 and the SOX9 target protein COL2 in monolayer-cultured FLCs. Multiple LMWF5A treatments of 3-D pellet FLC cultures over 2wks resulted in a significant decrease in IL-6 and significant increases in the major players of articular cartilage mechanics, aggrecan and highly-sulfated GAGs. CONCLUSIONS: These data support the hypothesis and clinical outcomes of two phase III clinical trials that LMWF5A-treatment induces chondrogenesis and supports functional cartilage. We propose that LMWF5A could maintain articular cartilage integrity in all joints following traumatic injury.
Authors: Gregory Thomas; Elizabeth Frederick; Melissa Hausburg; Laura Goldberg; Marshall Hoke; Michael Roshon; Charles Mains; David Bar-Or Journal: Patient Saf Surg Date: 2020-05-13
Authors: Gregory Thomas; Elizabeth Frederick; Lisa Thompson; Raphael Bar-Or; Yetti Mulugeta; Melissa Hausburg; Michael Roshon; Charles Mains; David Bar-Or Journal: J Transl Med Date: 2020-11-30 Impact factor: 5.531
Authors: David Bar-Or; Gregory Thomas; Leonard T Rael; Elizabeth Frederick; Melissa Hausburg; Raphael Bar-Or; Edward Brody Journal: Curr Rheumatol Rev Date: 2019