K. Lydon (Rochester, US)
Mayo ClinicPresenter Of 2 Presentations
24.1.8 - Exploration of Repopulation and Remodeling of Acellular Meniscus Grafts
Abstract
Purpose
Meniscus replacement using acellular scaffolds (CMI) and allografts (meniscus allograft transplantation) are established treatment options when meniscus repair is unfeasible. Outcomes show satisfying mid-to long-term pain relief while 70% of grafts show size reduction suggesting vitality issues such as incomplete integration in the host. Analysis of in vivo biopsies show evidence of cellular repopulation while it remains controversial to what extent. Further research is needed to elucidate meniscus repopulation and remodeling to enhance meniscus tissue regeneration. We investigated in-vitro scaffold repopulation and neo-tissue formation using a novel human tissue model. This “donut and hole” model allows for the analysis of specific repopulation features such as cell types and cell migration direction.
Methods and Materials
Human meniscus tissue from patients undergoing total knee arthroplasty, allografts (JRF Ortho®) and CMI (Stryker), were obtained, cut using an 8- and 4-mm circular biopsy punch and reconstructed according to Fig.1. Tissue constructs were cultured up to 40 days in DMEM and analyzed using LIVE/DEAD assay (ThermoFisher) and histology (SafraninO and H&E).
Results
At day 0 and 7, the presence of living cells was limited to the living meniscus samples. On day 14, living cells appear at the interface of the acellular allograft. On later timepoints, cells repopulate the surface of the allograft tissue, this was not observed for the CMI. On day 40, cells derived from the living meniscus tissue repopulated the superficial layers of the allografts while deep tissue repopulation was absent (Fig.1). Remarkably, tissue construct 1A showed earlier and more extensive repopulation in comparison with construct 1B while it is generally accepted that cells involved in meniscus graft repopulation originate from the adjacent synovium (Fig.2)(Arnocszky 1992).
Conclusion
Initial results show cellular ingrowth and time dependent repopulation of the frozen allograft which suggests this in-vitro model will allow us to investigate cellular repopulation and remodeling of acellular scaffolds and allografts.
P207 - Improving the Efficacy of Platelet Rich Plasma Through the Removal of Unwanted Cytokines
Abstract
Purpose
Platelet Rich Plasma (PRP) is commonly used to enhance the repair of musculoskeletal injuries due to its high concentration of platelets and proteins involved in the wound healing process. Due to variation in PRP preparation, and composition between patients, the clinical effectiveness of PRP treatment remains a controversy. Although PRP contains proteins beneficial to the wound healing process, it also contains a significant number of inflammatory molecules that could impair the wound healing process. This project aims to develop a microsphere technology that will specifically remove inflammatory cytokines from PRP, and to assess the changes in regenerative capacity of PRP after the removal of these cytokines. We hypothesize that the removal of these proteins will improve the effect of PRP on musculoskeletal tissue and enhance repair.
Methods and Materials
Polystyrene microspheres (Bang Laboratories) were functionalized with antibodies specific to inflammatory proteins (IL-1β and TNF-α), using carbodiimide coupling. Functionalized microspheres were then added to a single protein solution (1% BSA in PBS) or complex protein solution such as platelet lysate, spiked with target protein, and allowed to react for 1 hour at room temperature. Solutions were then centrifuged, supernatant was analyzed with an ELISA, and microspheres immunofluorescently stained to ensure proteins were bound to microspheres.
Results
15µg of antibody functionalized microspheres (MS) specifically bound and removed more than 1ng of target protein from both simple and complex solutions (Fig.1). Immunofluorescent imaging confirmed that proteins were bound to antibodies on the surface of the microspheres (Fig.2).
Conclusion
A technology has been developed to remove unwanted inflammatory cytokines from PRP. The results of this study confirmed the effectiveness of this technology, and the ability of the functionalized MS to specifically bind to target proteins in complex solutions such as PRP. Ongoing in vitro experiments are analyzing the effect of removing inflammatory proteins from PRP on musculoskeletal co-culture models.
Presenter Of 1 Presentation
P207 - Improving the Efficacy of Platelet Rich Plasma Through the Removal of Unwanted Cytokines
Abstract
Purpose
Platelet Rich Plasma (PRP) is commonly used to enhance the repair of musculoskeletal injuries due to its high concentration of platelets and proteins involved in the wound healing process. Due to variation in PRP preparation, and composition between patients, the clinical effectiveness of PRP treatment remains a controversy. Although PRP contains proteins beneficial to the wound healing process, it also contains a significant number of inflammatory molecules that could impair the wound healing process. This project aims to develop a microsphere technology that will specifically remove inflammatory cytokines from PRP, and to assess the changes in regenerative capacity of PRP after the removal of these cytokines. We hypothesize that the removal of these proteins will improve the effect of PRP on musculoskeletal tissue and enhance repair.
Methods and Materials
Polystyrene microspheres (Bang Laboratories) were functionalized with antibodies specific to inflammatory proteins (IL-1β and TNF-α), using carbodiimide coupling. Functionalized microspheres were then added to a single protein solution (1% BSA in PBS) or complex protein solution such as platelet lysate, spiked with target protein, and allowed to react for 1 hour at room temperature. Solutions were then centrifuged, supernatant was analyzed with an ELISA, and microspheres immunofluorescently stained to ensure proteins were bound to microspheres.
Results
15µg of antibody functionalized microspheres (MS) specifically bound and removed more than 1ng of target protein from both simple and complex solutions (Fig.1). Immunofluorescent imaging confirmed that proteins were bound to antibodies on the surface of the microspheres (Fig.2).
Conclusion
A technology has been developed to remove unwanted inflammatory cytokines from PRP. The results of this study confirmed the effectiveness of this technology, and the ability of the functionalized MS to specifically bind to target proteins in complex solutions such as PRP. Ongoing in vitro experiments are analyzing the effect of removing inflammatory proteins from PRP on musculoskeletal co-culture models.