L-PRP standardization and formation of semi-interpenetrating polymer networks of hyaluronic acid and fibrin form L-PRP for chondrogenic and osteogenic differentiation of human adipose-derived mesenchymal stem cells

Author(s)

Bruna Alice Gomes de Melo

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Engenharia Química
• Defense date: 2019-04-24

Examining board members

Maria Helena Andrade Santana; Angela Cristina Malheiros Luzo; William Dias Belangero; Bradley D Olsen; Maria Helena Ambrosio Zanin

• Advisor: Maria Helena Andrade Santana

Abstract

Leukocyte- and platelet rich plasma (L-PRP) is an autologous product, in which platelets, leukocytes, and other blood components are concentrated in a small fraction of plasma. Nowadays, concentrated of leukocytes and platelets are recognized as versatile biological products that are effective for tissues regeneration, presenting a wide range of clinical applications, and being largely used in the treatment of osteoarthritis. Hyaluronic acid (HA) is an endogenous polysaccharide present in the extracellular matrix (ECM) of articular cartilage and synovial fluid. The association between L-PRP and exogenous HA of different molar masses (MM) has shown synergistic benefits in the treatment of cartilage and bone injuries, promoting reduction of inflammation, pain relief and prolongation of the effects of the individual components. Despite the growing inclusion of HA associated with L-PRP in clinical protocols, there is a lack of information in the literature concerning the standardization of L-PRP and L-PRP-HA preparation. In addition, the involved mechanisms, formation of new structures and biological behavior resulted from this interaction lacks characterization. Therefore, this work aimed to standardize L-PRP preparation and study the structures formed from L-PRP-HA association, using HA of low and high MM (LMM and HMM, respectively). Their influence on soluble factors release was investigated, and on chondrogenic and osteogenic differentiation of human adipose-derived mesnchymal stem cells (h-AdMSCs) in vitro. By designing the whole blood centrifugation conditions, it was possible to obtain L-PRP with well-defined platelet/leukocyte and lymphocyte/granulocyte ratios that resulted in different biological behavior. The homogeneous and controlled mixture of L-PRP and HA formed fibrin-HA semi-interpenetrating polymer networks (semi-IPNs), after L-PRP activation by thrombin/calcium, with structure and rheological properties tunable with HA MM. The presence of HAs directly influenced the release of growth factors and cytokines by platelets and leukocytes present in the semi-IPNs, as well as the adhesion, growth and chondrogenic and osteogenic differentiation of h-AdMSCs. These results are relevant to standardize the preparation of L-PRP, L-PRP-HA mixture and fibrin-HA hydrogels, aiming to improve clinical protocols. In addition, this work contributes to the understanding of the phenomena involved in the L-PRP-HA association that lead to tissues regeneration (AU)

• FAPESP's process: 15/23134-8 - Influence of the association between platelet-rich plasma and hyaluronic acid on the release of growth factors, cellular proliferation and osteogenic/ chondrogenic differentiation
• Grantee: Bruna Alice Gomes de Melo
• Support Opportunities: Scholarships in Brazil - Doctorate