Human Chondrocyte Response Cultured on Pre-treated PLGA Mesh Scaffold using Platelet-rich Plasma

INTRODUCTION: The meniscal lesions occurring in the non-vascularized inner twothirds heal poorly or not at all. A cell-based tissue-engineered construct can be employed for treating these intractable lesions [1-4]. Woven Vicryl mesh (PLGA, Ethicon) is a biocompatible and biodegradable synthetic polymer used clinically. This FDA approved material can be employed as a scaffold for meniscal repair because it is thin and flexible enough to interpose into meniscal tear lesions. Recently, we developed a dynamic cell-seeding technique and demonstrated the healing capacity of the cell-seeded scaffolds in a novel in vivo meniscus repair mouse model [2, 3, 4]. Nevertheless, utilized as a scaffold, it should provide enhanced cell attachment and uniform cell distribution that will result in optimal tissue repair and regeneration once implanted in vivo. Platelet-rich plasma (PRP) may prove to be a good candidate substance for biomaterial surface treatment because it has various growth factors and roles as a raw material for bio-film formation [5]. In a previous study, we developed a novel surface modification technique for the PLGA mesh scaffold using centrifugal PRP treatment combined with calcium addition. We hypothesized that the PLGA scaffold modification using PRP pretreatment would provide a more favorable biomimetic and biocompatible surface for cell adhesion, survival, and proliferation. In this study, we evaluated human chondrocytes cultured on PLGA scaffold underwent PRP pretreatment. METHODS: PRP and scaffold preparation: Allogeneic leukocyte-depleted PRP was obtained from our hospital blood bank. The mean platelet count in the PRP ranged from 800/nl to 1,100/nl. PRP of 5 donors was mixed and used for the experiments. The woven PLGA mesh scaffold (Vicryl, Ethicon) measuring 20×8 mm (thickness, 0.2 mm) was prepared. Centrifugal PRP treatment: The scaffolds were inserted into the bottom of 6 well plates and immersed into 1,000 μl of PRP. The plates were centrifuged at 150g for 10 min and incubated at 37 °C at an oscillation rate of 70 Hz for 60 min. Then, the scaffold was flipped 180° and the same procedure was done for the other side of scaffolds. After washing three times with DPBS (Dulbecco’s phosphate-buffered saline), the scaffolds were soaked into 1,000 μl of DMEM media at 37 °C for 10 min. The calcium in the media initiated the clotting cascade to form a fibrin network. Human chondrocyte isolation and seeding: Human articular chondrocytes were isolated from femoral head cartilage of 10 patients that underwent total hip arthroplasty after informed consent and approval of our IRB. At 80-90 % confluency after the first passage, the chondrocytes (P1) were harvested, mixed, and seeded to the scaffold. The scaffolds were placed in 15 mL polypropylene tubes containing 10 million chondrocytes in 5 mL of culture medium. The tubes were placed horizontally into a bioreactor at 37 °C at an oscillation rate of 40 Hz for 7 days [4]. Cell response evaluations: Cell distribution and viability on the scaffold was observed with a live/dead viability assay. Cell number for each cell-seeded scaffold was determined using a cell counting kit (CCK-8). A chondrocyte specific gene expression was determined using a RT-PCR. The microstructural changes were observed with a scanning electron microscopy (SEM). The chondrocytes cultured on non-treated scaffolds were used as a control. Statistics: The data was presented as