1. Project Timeline
Working Schedule
Project Initiation Date: 15/11/2022
Project End Date: 15/11/2024
2. Introduction
Osteoarthritis (OA) is a common joint disease affecting over 500 million people worldwide, characterized by degenerative changes in joint cartilage, resulting in symptoms such as pain and restricted mobility. While treatment primarily involves symptom-relieving medications and physical therapy, interest in platelet-rich plasma (PRP) applications targeting tissue regeneration has grown in recent years. PRP is a blood component prepared by centrifuging a patient’s own blood to concentrate platelets above basal levels. Different PRP formulations vary based on the amount of platelets and growth factors they contain. This study aimed to compare the metabolomic profiles of various PRP formulations.
The study found significant metabolic differences among PRP types and showed that storage conditions affect their biological activities. Pathway analyses revealed that PRP types possess distinct biochemical profiles related to energy metabolism, lipid biosynthesis, and oxidative stress management.
It was concluded that developing application strategies tailored to the unique characteristics of each PRP formulation and improving storage protocols have a decisive impact on clinical outcomes. These findings provide a crucial foundation for enhancing the clinical potential of PRP and guiding personalized treatment approaches. Further analyses at the cellular level and future in vivo applications will enable a deeper understanding of the biological and clinical effects of PRP.
3. Aim and goals
The aims of this project are to analyze the metabolomic characteristics and biological effects of L-PRP, P-PRP, PRF, and PRGF, with a specific focus on their role in osteoarthritis treatment. The key objectives include:
- Identifying similarities and differences in the metabolomic profiles of L-PRP, P-PRP, PRF, and PRGF.
- Determining potential metabolomic profile changes after freezing and thawing to evaluate the stability of PRP formulations under these conditions.
- Investigating the effects of PRP formulations on chondrocytes by conducting metabolomic pathway analyses on cell lysates obtained from co-culture experiments with normal and osteoarthritic (OA) chondrocytes.
Goals of the Project
- To characterize the metabolomic differences between L-PRP, P-PRP, PRF, and PRGF.
- To assess the impact of freezing and thawing on PRP stability using metabolomic analyses.
- To determine the distinct metabolic effects of PRP formulations on normal and OA chondrocytes through metabolomic pathway analysis.
The ultimate goal of this project is to identify metabolites that contribute to the preservation of chondrocytes under physiological conditions and stimulate their regeneration under pathological conditions. These findings will serve as a foundation for future studies aimed at improving cartilage repair and osteoarthritis treatment.
- Work Packages
WP1 – PRP Preparation, Characterization, and Storage
This work package focuses on the preparation, characterization, and storage (both fresh and frozen) of different PRP formulations, including L-PRP, P-PRP, PRF, and PRGF. Pre- and post-preparation cell counts are conducted to ensure the suitability of samples for metabolomic analysis.
1.1. Blood Collection and PRP Preparation
1.2. Cell Counting in Blood and PRP Samples
1.3. Division and Transfer of PRP Samples to the Laboratory for Fresh and Frozen Storage
WP2 – Metabolomic Analysis of PRP and Evaluation of Freeze-Thaw Stability
This work package aims to analyze PRP at the metabolomic level and compare fresh and frozen PRP formulations to assess potential changes due to freeze-thaw cycles. A platform capable of analyzing 600 metabolites was established, and both targeted and untargeted metabolomic analyses were conducted using Q-TOF LC/MS.
2.1. Establishment of a Platform for the Identification of 600 Metabolites Using a Metabolite Standard Kit
2.2. Untargeted Metabolomic Analysis Using Q-TOF LC/MS
2.3. Targeted Metabolomic Analysis Using Q-TOF LC/MS
2.4. Stability Studies
WP3 – Co-Culture of PRP with OA and Healthy Chondrocytes and Evaluation of Its Effects
This work package focuses on culturing OA and healthy chondrocytes, co-culturing them with different PRP formulations, and determining IC50/ED50 values. The effects of PRP on autophagy, apoptosis, and extracellular matrix (ECM) production were examined using immunofluorescence, flow cytometry, and immunohistochemistry techniques.
3.1. Real-Time Cell Proliferation Analysis
3.2. Immunofluorescence
3.3. Flow Cytometry
3.4. Immunohistochemistry
3.5. ELISA
WP4 – Metabolomic Evaluation of PRP’s Effects on OA and Healthy Chondrocytes
The primary goal of this work package is to analyze the metabolomic impact of PRP on OA and healthy chondrocytes. Metabolomic analysis results were evaluated using a systems biology approach, integrating findings with WP3.
4.1. Untargeted Metabolomic Analysis Using Q-TOF LC/MS
4.2. Targeted Metabolomic Analysis Using Q-TOF LC/MS
4.3. Proteomic Analysis Using Q-TOF LC/MS
4.4. Evaluation of Molecular Effects Using a Systems Biology Approach (Comparison with WP3 Data)
Conclusion:
As a result, the project has established a new standard in the metabolomic profiling of PRP formulations, providing a detailed characterization of their biological differences. These findings serve as a crucial foundation for enhancing the clinical potential of PRP and guiding personalized treatment approaches. Further cellular-level analyses and future in vivo applications will enable a deeper understanding of PRP’s biological and clinical effects.