The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon

Abstract

Objectives: Platelet-rich fibrin matrix (PRFM) has been proved to enhance tenocyte proliferation but has mixed results when used during rotator cuff repair. The optimal PRFM preparation protocol should be determined before clinical application. To screen the best PRFM to each individual's tenocytes effectively, small-diameter culture wells should be used to increase variables. The gelling effect of PRFM will occur when small-diameter culture wells are used. A co-culture device should be designed to avoid this effect.

Methods: Tenocytes harvested during rotator cuff repair and blood from a healthy volunteer were used. Tenocytes were seeded in 96-, 24-, 12-, and six-well plates and co-culture devices. Appropriate volumes of PRFM, according to the surface area of each culture well, were treated with tenocytes for seven days. The co-culture device was designed to avoid the gelling effect that occurred in the small-diameter culture well. Cell proliferation was analyzed by water soluble tetrazolium-1 (WST-1) bioassay.

Results: The relative quantification (condition/control) of WST-1 assay on day seven revealed a significant decrease in tenocyte proliferation in small-diameter culture wells (96 and 24 wells) due to the gelling effect. PRFM in large-diameter culture wells (12 and six wells) and co-culture systems induced a significant increase in tenocyte proliferation compared with the control group. The gelling effect of PRFM was avoided by the co-culture device.

Conclusion: When PRFM and tenocytes are cultured in small-diameter culture wells, the gelling effect will occur and make screening of personalized best-fit PRFM difficult. This effect can be avoided with the co-culture device.Cite this article: C-H. Chiu, P. Chen, W-L. Yeh, A. C-Y. Chen, Y-S. Chan, K-Y. Hsu, K-F. Lei. The gelling effect of platelet-rich fibrin matrix when exposed to human tenocytes from the rotator cuff in small-diameter culture wells and the design of a co-culture device to overcome this phenomenon. Bone Joint Res 2019;8:216-223. DOI: 10.1302/2046-3758.85.BJR-2018-0258.R1.

Keywords: Co-culture; Gelling effect; Platelet-rich fibrin matrix; Rotator cuff tears; Tenocytes.

Fig. 1

The concept of a co-culture device to avoid the gelling effect. Top left) cell seeding in the cell chamber. Top right) platelet-rich fibrin matrix (PRFM) added into another chamber (PRFM chamber) without cell seeding. PRFM gelling may not affect the cell in the chamber because of the polydimethylsiloxane (PDMS) barrier. Bottom right) add culture medium into the PRFM chamber until it crosses over the PDMS barrier. Bottom left) growth factors in PRFM with lighter gravity dispersed in the culture medium are disseminated to the cell chamber to stimulate tenocyte proliferation. GF, growth factor.

Fig. 2

The co-culture device design. a) Tenocytes seeded in the cell chamber. b) Platelet-rich fibrin matrix (PRFM) was added into the PRFM chamber to avoid the gelling effect. c) Culture medium (arrow) was added into the PRFM chamber until the fluid level crossed over the separation polydimethylsiloxane (PDMS) barrier, causing growth factors dispersed in the culture medium to be disseminated to the cell chamber to stimulate tenocyte proliferation.

Fig. 3

The gelling effect (arrow) of platelet-rich fibrin matrix (PRFM) occurred in the 96-well plate.

Fig. 4

Tenocytes treated with the same and different volumes of platelet-rich fibrin matrix (PRFM). a) Tenocytes treated with 10 µl PRFM in different culture wells. The PRFM gelling effect was observed in small-diameter culture wells (96- and 24-well plates) but not in large-diameter culture wells (12- and six-well plates) or co-culture devices. There was a significant decrease in tenocyte proliferation in small-diameter culture wells. b) Tenocytes treated with different volumes of PRFM according to the surface area of each culture well. The PRFM gelling effect was observed in small-diameter culture wells (96- and 24-well plates) but not in large diameter culture wells (12- and six-well plates) and co-culture device. There was a significant decrease in tenocyte proliferation in small-diameter culture wells and a significant increase in tenocyte proliferation in the six-well plate and co-culture device. *p-value < 0.05.

Fig. 5

Platelet-rich fibrin matrix (PRFM) in the conventional culture well and co-culture device on day seven. a) Platelet aggregates (arrow) were found in the conventional 96-well plate. Decreased tenocyte proliferation was caused by the gelling effect of PRFM due to direct contact of tenocytes and PRFM. b) The PRFM gelling effect was avoided by the use of the co-culture device. Tenocytes (arrow) were more organized in the co-culture device than in the conventional 96-well plate.