Comparison of Cryopreserved Amniotic Membrane and Umbilical Cord Tissues for use in Foot and Ankle Reconstructive Procedures

Transcription

1 Comparison of Cryopreserved Amniotic Membrane and Umbilical Cord Tissues for use in Foot and Ankle Reconstructive Procedures Howard M. Kimmel 1, Ek Kia Tan 2, Hua He 2, Julie O Connell 3 1 Buckeye Foot Care, Brook Park OH 2 TissueTech, Inc., Miami, FL 3 Amniox Medical, Inc., Atlanta, GA

2 Disclosure Kimmel HM Consultant for Amniox Medical Inc. Tan EK Employee of TissueTech Inc. He H Employee of TissueTech Inc. O Connell J Employee of Amniox Medical Inc.

3 Background Fetal tissue derived products have demonstrated success when used clinically as adhesion barriers during foot and ankle reconstructive procedures Many fetal tissue products consist of the amniotic membrane derived from the amniotic sac of the placenta proper, with some including the underlying chorion Currently, only one fetal tissue product utilizes amniotic membrane derived from the umbilical cord The purpose of this study was to characterize key structural, biochemical, and functional differences between cryopreserved amniotic membrane and cryopreserved umbilical cord tissues that are necessary for their therapeutic potential

4 Materials & Methods Materials: Cryopreserved amniotic membrane (; CLARIX TM 100) and cryopreserved umbilical cord (; CLARIX TM 1K, Amniox Medical Inc., Atlanta, GA) Methods: Histology: Samples were stained for hematoxylin and eosin, Masson s trichrome, and Safranin-O Histochemistry: Tissue matrix hyaluronic acid (HA) content was measured using HA binding protein (HABP) fluorescent histochemistry Macrophage functional assessment: RAW264.7 macrophage proliferation and cell death was assessed in cultures on cryopreserved and tissues Inflammatory cytokine levels: Secreted levels of the proinflammatory cytokine, IL-12, and the anti-inflammatory cytokine, IL-10, were measured in RAW264.7 cells cultured on cryopreserved and tissues

5 Results A B H&E HAase (-) C D MAS SafO E F HAase (+) Collagen, non-sulfated glycosaminoglycans, and HA content were similarly distributed across the matrix. However, the additional Wharton s Jelly matrix considerably increased the amount of all ECM components

6 Results HAase(-) HAase(+) 5 6 Total HA/Weight Ratio HA/Protein Ratio HMW HA I II LMW HA III Cryopreserved umbilical cord contains approximately 10-fold higher levels of HA compared to amniotic membrane

7 Results Cryopreserved and significantly reduced RAW264.7 macrophage cell proliferation compared to control Cryopreserved significantly increased macrophage cell death compared to and control BrdU Proliferation/Protein Cell Death/Protein

8 Results IL-10/Protein IL-12/Protein IL-10/IL-12 Ratio CTL CTL Secreted levels of the pro-inflammatory cytokine IL-12 were significantly reduced in and tissues compared to control. Cryopreserved further decreased IL-12 levels compared to. Secreted levels of IL-10, an anti-inflammatory cytokine were increased in tissues compared to both control and tissues CTL

9 Discussion Cryopreserved umbilical cord tissues contain significantly higher amounts of extracellular matrix components, including high molecular weight hyaluronic acid, that are attributed to the antiinflammatory and anti-scarring properties of fetal tissues that aid in tissue healing Cryopreserved umbilical cord outperformed amniotic membrane in anti-inflammatory functional assays using RAW264.7 macrophages by reducing macrophage proliferation and increasing macrophage cell death compared to amniotic membrane alone Levels of pro-inflammatory cytokines are significantly decreased while levels of anti-inflammatory cytokines are increased in cryopreserved- compared to cryopreserved-

10 Conclusions These results indicate that cryopreserved umbilical cord may offer physiological advantages within the wound environment, ultimately translating to greater clinical efficacy compared to amniotic membrane alone

11 References 1. Tseng SCG, Espana EM, Kawakita T, Di Pascuale M a, Li W, He H, et al. How does amniotic membrane work? Ocul Surf Jul;2(3): Dua HS, Gomes JAP, King AJ, Maharajan VS. The amniotic membrane in ophthalmology. Surv Ophthalmol. 49(1): Bouchard CS, John T. Amniotic membrane transplantation in the management of severe ocular surface disease: indications and outcomes. Ocul Surf Jul;2(3): He H, Li W, Chen S-Y, Zhang S, Chen Y-T, Hayashida Y, et al. Suppression of activation and induction of apoptosis in RAW264.7 cells by amniotic membrane extract. Invest Ophthalmol Vis Sci Oct;49(10): He H, Li W, Tseng DY, Zhang S, Chen S-Y, Day AJ, et al. Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-alpha-inhibitor (HC*HA) purified from extracts of human amniotic membrane. J Biol Chem Jul 24;284(30): Zhang S, He H, Day AJ, Tseng SCG. Constitutive expression of inter-α-inhibitor (IαI) family proteins and tumor necrosis factor-stimulated gene-6 (TSG-6) by human amniotic membrane epithelial and stromal cells supporting formation of the heavy chain-hyaluronan (HC-HA) complex. J Biol Chem Apr 6;287(15): He H, Zhang S, Tighe S, Son J, Tseng SCG. Immobilized heavy chain-hyaluronic acid polarizes lipopolysaccharide-activated macrophages toward M2 phenotype. J Biol Chem Sep 6;288(36): Adzick NS, Lorenz HP. Cells, matrix, growth factors, and the surgeon. The biology of scarless fetal wound repair. Ann Surg Jul;220(1):10 8.