Antibacterial Potential of Platelet Lysate Membranes for Orthopedic Applications

Statement of Purpose: Microbial infections are one of the most serious complications on post-transplant and surgery. The recurrent use of antibiotic therapeutic to treat such infections has led to the increase of microbial strain resistance, and thus, reducing antibiotic efficacy^1. Platelet-rich hemoderivatives (PRHd) gained great interest in the medical field, namely in sports medicine and orthopaedics^2,3. Platelet lysate (PL), a PRHd produced by cryogenic disruption of platelet concentrates, has been proposed as an alternative source of growth factors. PL membranes were previously developed in our group as prospective bioinstructive patches for tendon regeneration under the hypothesis that tendon cells respond positively to PL-derived biochemical signals. The present study focused on assessing the antibacterial potential of these PL membranes given that previous studies suggested that platelet-rich plasma (PRP) presents antimicrobial properties. For instance, PRP activation through thrombin exhibited antimicrobial properties in vitro⁴, but the antibacterial properties of PL proteins immobilized as crosslinked membranes are still unknown. Thus, this study aimed at investigating the potential of PL membranes as antibacterial surfaces for biomedical implants by studying their effects against Staphylococcus aureus (Gram positive), a common orthopaedic surgical site infection pathogen.

Methods: PL membranes were fabricated as previously described⁵ and characterized in terms of degradation and protein release profile. Human tendon-derived cells (hTDCs) were seeded on PL membranes and their behavior was investigated regarding proliferation, metabolic activity and extracellular matrix (ECM) production up to 21 days in culture. The antibacterial properties of PL membranes were evaluated against S. aureus ATCC 29213. PDMS films were used as controls. The number of viable counts of S. aureus was assessed up to 72h growth period by live/dead staining. Biofilm formation and distribution were analysed by scanning electron microscopy (SEM). Each experiment was performed in triplicate. Statistical analysis was performed using a one-way ANOVA test, differences were considered statistically significant whenever p<0.05.

Results: Overall, results from the present work showed that PL membranes remained stable for up to 30 days in PBS and that the release profile of PL-derived proteins followed a typical controlled release pattern (Fig. 1A), as described by Babo et al⁵. Regarding biological performance, cell content of hTDCs seeded in PL-membranes was maintained over time, possibly avoiding an extensive proliferative phase, which leads to scar formation in vivo during tendon repair. Cells deposited tendon-related ECM proteins, including collagen types I and III, and tenascin-C.

The number of viable counts of S. aureus significantly decreased after 24h of incubation when compared to the PDMS control (Fig. 1B-D). Bacteria adherence and biofilm formation significantly decreased after 24h incubation when compared to the PDMS control. Likewise, the formation of bacterial biofilm was only observed in the PDMS controls, and not in PL membranes after 72 h incubation. Results suggest that PL membranes have an antibacterial effect, by preventing the adherence and proliferation of S. aureus, as well as the formation of biofilm.

Conclusions: PL membranes were fabricated through a very simple and reproducible method by crosslinking PL proteins. The outcomes of this study suggest that PL membranes inhibited the formation of bacterial biofilm and adherence to the surface of the membrane. These membranes can modulate cellular activity in situ, actingas a reservoir of bioactive molecules derived from PL, which supports their application as bioinstructive and protective patches for tendon regeneration. 

References: 

1. Berkner S. EMBO Reports. 2014;15:740-744.

2. Li H. Adv Health Mater. 2013; 2:1277-1284

3. Intini G. Biomaterials. 2009;30:4956–4966.

4. Drago L. BMC Microbiology. 2013;13:47-52.

5. Babo P. Inflamm Regen. 2014; 34:33-44.

Acknowledgements: The authors thank FCT–Fundação para a Ciência e a Tecnologia in the framework of FCT-POPH-FSE, RC-A PhD grant SFRH/BD/96593/2013, ARF Post-Doc grant SFRH/BPD/100760/2014, and MEG grant IF/00685/2012.