Poster Abstracts
John Shou, PhD
Principal Investigator and Chief Scientific Officer
Regenative Labs
Pensacola, Florida
Naomi Lambert, BS
Clinical Site Coordinator
Regenative Labs
Pensacola, Florida
Three-Dimensional Electron Microscopy of Human Umbilical Cord Tissue Allograft Pre and Post Processing: A Literature Comparison In an ISO class 5 biologic safety cabinet, the umbilical cord was rinsed with saline to remove excess blood residue and clots. Various sized cross-sections of the umbilical cord were cut and placed on a sterile drying tray, then desiccated in a high nitrogen concentration drying chamber. WJ was aseptically dissociated from the rinsed umbilical cord. After dissociation, 150 mg of tissue was suspended in approximately 2mL of sterile Sodium Chloride 0.9% solution. The sample was not combined with cells, tissues, or articles other than the exceptions outlined in 21 CFR Part 1271.10(a) (3) (HCT/P Regulation). Pre-processed and post-processed desiccated tissue samples were received by the University of Montana laboratory for analysis and electron microscope imaging. The tissue samples were transferred to a sticky carbon surface and coated with a thin layer of iridium to mitigate charging in the microscope.
Background: One in four adults in the US suffers from cartilage degeneration of the Intervertebral Disc (DDD) or load-bearing joints (DJD). Since cartilage is avascular, it has a limited regenerative capacity. Conventional non-surgical treatments provide brief symptomatic relief, have side effects, and do not address the cartilage defect. Perinatal birth tissue allografts are a novel frontier for bio-mechanical cartilage engineering research. The tissues of interest include umbilical cord-derived Wharton’s Jelly (WJ). Since its initial discovery, there has been significant interest in its use for regenerative medicine applications [2]. WJ spans the entire length of the umbilical cord, providing protection, cushioning, and structural support to the encased vessels [2,3]. Initial research centered on WJ as a cellular product dependent on the metabolic activity of living cells [3]. However, classified as a loose connective tissue, WJ exerts an effect independent of cellular activity [3]. WJ functions as an ideal structural system to transplant chemokine and growth factors, with its biomechanical microarchitecture for collagen extracellular matrix formation in collagen-based defects [4].
Purpose/Objectives: Regeneration of collagen structural tissue defect occurs as ECM is regenerated in the base of the defect, restoring the original relevant characteristics of the damaged tissue [10]. Scar tissue will only achieve a maximum of 80% strength of the native tissue [11]. There is great potential for the WJ matrix derived from the human Umbilical Cord (UC) to act as an ECM scaffold and augment the regenerative process [10]. WJ contains an abundant amount of collagen and hyaluronic acid, growth factors, and proteoglycans, essential components of ECM [2,10]. Since WJ is a comprehensive source of human extracellular matrix proteins and growth factors; there may be numerous clinical applications for collagen-based structural tissue defects [3]. This study aimed to assess WJ tissue samples via a ZEISS Supra 55VP Field-Emission Scanning Electron Microscope (SEM) at 100 and 300 nm resolution scales.
Method: Pre-processed umbilical cord tissue samples
Processed umbilical cord tissue samples product
Imaging
Results: The present study reports the anatomic structural compatibility of a human Umbilical Cord Tissue (UCT) allograft before and after processing to provide objective evidence of minimal manipulation. The resultant comparison establishes examples of homologous use with objective evidence. The SEM images of the post-processed samples reveal that the structural integrity of collagen fibers within the ECM is maintained (Figure 2). The diameter of the UCT fibers increased from approximately 61nm pre-processing to 65 nm post-processing. This increase could be due to slight swelling from the saline rinse and is not statistically significant.
Conclusions: The multidirectional collagen network observed in WJ appears to be structurally comparative to the collagenic organization found in the extracellular matrix of cartilage [16,19]. Published research indicates that WJ may demonstrate chondrogenic differentiation capabilities, which could be beneficial in cartilage regeneration [19]. The anatomic structural similarities between different collagen networks indicate that human umbilical cord tissue allografts are homologous in structure to the extracellular matrix found in cartilage and dermis. The breakdown of collagen results in the deterioration of cartilage [8]. While applications of human umbilical cord tissue allografts have been used extensively in the clinical treatment of cartilage tears, there is also potential for allografts to be used in the pathology of intervertebral discs, muscle tears, and deep tunneling wounds [20]. As a nonsurgical option, applying WJ allografts to structural tissue defects warrants clinical consideration. Despite WJ being a non-vascular, there is also the promise of homologous applications for WJ in vascularized tissue. In figure 6C, the organization of the collagen fibril network in skeletal muscle closely resembles the multidirectional collagen fibers seen in the post-processed umbilical cord tissue samples. The clinical applications of WJ allografts may be profound in both vascularized and non-vascularized structural defects. Umbilical cord tissue allografts in regenerative medicine will be continually enhanced with additional discoveries of homologous use applications in clinical research data.
References: 2. Gupta A, El-Amin SF 3rd, Levy HJ, Sze-Tu R, Ibim SE, Maffulli N. Umbilical cord-derived Wharton's jelly for regenerative medicine applications. J Orthop Surg Res. 2020 Feb 13;15(1):49. doi: 10.1186/s13018-020-1553-7. PMID: 32054483; PMCID: PMC7017504.
3. Deus IA, Mano JF, Custódio CA. Perinatal tissues and cells in tissue engineering and regenerative medicine. Acta Biomater. 2020 Jul 1;110:1-14. doi: 10.1016/j.actbio.2020.04.035. Epub 2020 May 14. PMID: 32418650.
4. Jadalannagari S, Converse G, McFall C, Buse E, Filla M, Villar MT, Artigues A, Mellot AJ, Wang J, Detamore MS, Hopkins RA, Aljitawi OS. Decellularized Wharton's Jelly from human umbilical cord as a novel 3D scaffolding material for tissue engineering applications. PLoS One. 2017 Feb 21;12(2):e0172098. doi: 10.1371/journal.pone.0172098. Erratum in: PLoS One. 2017 Mar 7;12 (3):e0173827. PMID: 28222169; PMCID: PMC5319682.
10. Penolazzi L, Pozzobon M, Bergamin LS, D'Agostino S, Francescato R, Bonaccorsi G, De Bonis P, Cavallo M, Lambertini E, Piva R. Extracellular Matrix From Decellularized Wharton's Jelly Improves the Behavior of Cells From Degenerated Intervertebral Disc. Front Bioeng Biotechnol. 2020 Mar 27;8:262. doi: 10.3389/fbioe.2020.00262. PMID: 32292779; PMCID: PMC7118204.
11. Xue M, Jackson CJ. Extracellular Matrix Reorganization During Wound Healing and Its Impact on Abnormal Scarring. Adv Wound Care (New Rochelle). 2015 Mar 1;4(3):119-136. doi: 10.1089/wound.2013.0485. PMID: 25785236; PMCID: PMC4352699.
16. Zhang Y, Liu S, Guo W, Wang M, Hao C, Gao S, Zhang X, Li X, Chen M, Jing X, Wang Z, Peng J, Lu S, Guo Q. Human umbilical cord Wharton's jelly mesenchymal stem cells combined with an acellular cartilage extracellular matrix scaffold improve cartilage repair compared with microfracture in a caprine model. Osteoarthritis Cartilage. 2018 Jul;26(7):954-965. doi: 10.1016/j.joca.2018.01.019. Epub 2018 Jan 31. PMID: 29391278.
19. Majore I, Moretti P, Stahl F, Hass R, Kasper C. Growth and differentiation properties of mesenchymal stromal cell populations derived from whole human umbilical cord. Stem Cell Rev Rep. 2011 Mar;7(1):17-31. doi: 10.1007/s12015-010-9165-y. PMID: 20596801.
20. Sadlik B, Jaroslawski G, Puszkarz M, Blasiak A, Oldak T, Gladysz D, Whyte GP. Cartilage Repair in the Knee Using Umbilical Cord Wharton's Jelly-Derived Mesenchymal Stem Cells Embedded Onto Collagen Scaffolding and Implanted Under Dry Arthroscopy. Arthrosc Tech. 2017 Dec 25;7(1):e57-e63. doi: 10.1016/j.eats.2017.08.055. PMID: 29552470; PMCID: PMC5852271.