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Introduction: Standard surgical procedures such as debridement and microfracture in combination with application of various biomaterials into defect were suggested to support cartilage healing. Although these techniques provide short-term relieve from pain and temporary restoration of joint function, long-term studies discovered that healing process led to formation of fibrocartilage with high content of COL1 instead of COL2. Therefore, the most important improvement that TERM approaches should bring to cartilage treatment is to support formation of hyaline cartilage with high content of COL2.
Hyaluronic acid is a natural polymer which is often used for treatment of cartilage defects. Tyraminated derivative of hyaluronic acid (HA-TA) was proved to be a suitable material for preparation of in situ gellable hydrogels which enable cell encapsulation [1,2]. HA-TA-based scaffolds support chondrogenic differentiation of surrounding cells but don´t support cell attachment. On that account, combination of these scaffolds with patient´s own platelet-rich plasma (PRP) is gaining attention. Plasma proteins serve as a source of adhesion molecules; cytokines and growth factors participate in directing cell differentiation.
Here we present a fully biocompatible photocrosslinkable HA-TA-based hydrogel which allows combination with PRP plasma and can be applied during arthroscopic surgery and solidify in situ after exposure to arthroscope light. Initiation of photocrosslinking reaction by visible light is enabled by photosensitizer riboflavin.
Methodology: Influence of HA-TA concentration (2 % or 3 % (w/v)) and PRP concentration (12,5 %, 25 % or 50 % (v/v)) in hydrogel on cell growth and differentiation was tested in vitro.
HA-TA was dissolved in riboflavin solution overnight, sterilized in autoclave and mixed with human PRP. This mixture was injected in culture wells and solidified by irradiation by arthroscope light for 3 min. Chondrocytes were seeded on top of hydrogel layers and cultured in standard culture medium for 14 days. Cell proliferation and gene expression were assessed. Hydrogels were characterized by determination of viscoelastic properties and swelling ratio.
Results: Both 2 % and 3 % HA-TA hydrogels containing PRP supported chondrocyte attachment and increased COL2/COL1 ratio compared to control, but hydrogel with higher HA-TA content was less stable due to higher swelling.
Concentration of PRP in 2 % HA-TA hydrogels influenced cell growth and gene expression: cell attachment, proliferation and production of COL1 were highest on hydrogels containing 50 % PRP and lowest on hydrogels with 12,5 % PRP. Production of chondrogenic differentiation-related genes COL2, SOX9 and ACAN was highest on hydrogels containing 25 % PRP.
Therefore, 2 % HA-TA and 25 % PRP appears to be the optimal composition of hydrogel which supports both cell growth and chondrogenic phenotype.
Conclusion: We developed a photocrosslinkable HA-TA-based material for treatment of cartilage defects which can be applied during arthroscopic surgery. Using riboflavin as a photosensitizer allows in situ solidification of the material after irradiation by arthroscope light. Developed material allows combination with PRP plasma and supports cell adhesion and chondrogenic differentiation.
References:
1. Bystronova, J. et al., RSC Adv. 8, 7606-7614 (2018).
2. Gallagher, L. B. et al., Acta Biomater. 107, 78-90 (2020).
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