Human meniscal proteoglycan metabolism in long-term tissue culture

Abstract

For the purpose of human meniscal allografting, menisci have been maintained viable in in vitro culture. The influence of long-term tissue culture on the extracellular matrix metabolism of the meniscus has been studied. Fetal calf serum (FCS) was used as a supplement for the growth factors necessary to maintain optimal meniscal cell metabolism. A series of semilunar cartilage samples was cultured under serum-free conditions since foreign proteins could be responsible for immunological problems after eventual allografting. The proteoglycan metabolism in human menisci cultured in FCS-supplemented and in serum-free culture media was compared. To rule out any influence of topographical variations in glycosaminoglycan (GAG) content on proteoglycan (PG) metabolism, GAG concentrations within the tissue were determined, and sulphate (35S) incorporation was studied in tissue samples with a comparable biochemical composition. Sulphate incorporation was preserved when 20% FCS was added to the nutrient medium. The meniscal tissue fibroblasts continued to produce 35S-PG during 4 weeks of culture. The PG molecules were shown to consist of PG-aggregates, monomers and a low molecular-weight PG population. Newly synthesized GAG consisted of approximately 55% chondroitin 4- and 6-sulphate and 33% dermatan sulphate. In the presence of serum, 35S incorporation in PG and in the PG-aggregate fraction significantly increased during the first 2 weeks and then decreased during the following 2 weeks of in vitro culture. Newly synthesized PG-aggregates were almost entirely accumulated in the tissue during these weeks. In the 3rd week the values for this parameter decreased slightly. 35S-PG synthesis dramatically declined after 4 weeks of in vitro culture. Catabolism probably resulted in increased proportions of 35S-PG in the incubation media. In the absence of serum, 35S-PG production also increased in the 2nd week of culture. However, 35S activity was almost exclusively found in small PG, and this material apparently diffused to the incubation media. Consequently, catabolism is higher, and the immobilization of 35S-PG is poor when FCS is not added to the culture media. Our findings suggest that menisci are maintained in viable condition and may serve for allografting at least during 2 weeks of tissue culture.

Transplantation of Viable Meniscal Allograft

“…The allograft was maintained in culture for two weeks in Dulbecco Modified Eagle Medium (DMEM; Gibco Invitrogen, Merelbeke, Belgium) supplemented with 20% autologous serum prior to transplantation. Previous studies have demonstrated that meniscal cells remain viable and continue to synthesize their extracellular matrix molecules in this culture system 14 . During the culture period, there was ample time to screen the donors for transmissible diseases.…”

“…Cultured menisci have been shown to remain viable and to continue to produce extracellular matrix compounds⎯hence, the term "viable meniscal allograft." 14 After a two-week culture period, the meniscus is transplanted into the recipient knee. The proportion of cells .…”

rAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and α-SMA expression in human meniscal lesions

“…The proteoglycan contents were measured by binding to the dimethylmethylene blue dye. 44,47,59 The DNA contents were determined with a fluorimetric assay using Hoechst 33258. 24,27,44,47 Measurements were performed using a GENios spectrophotometer/fluorometer (Tecan Deutschland GmbH, Crailsheim, Germany).…”

Experimental research Assessment of apoptosis, MMP-1, MMP-3, TIMP-2 expression and mechanical and biochemical properties of the fresh rabbit’s medial meniscus stored two weeks under tissue culture condition

“…Verbruggen et al . claim that the viability of stored meniscal cells should be maintained through their ability to produce matrix particles [6]. Le Graverand et al .…”

“…Verbruggen et al . demonstrated that fresh meniscal cells may be stored under tissue culture conditions for 2 weeks while maintaining their viability, and their ability to produce extracellular matrix elements [6]. Fresh menisci stored under tissue culture conditions are used in clinical practice, and Verdonk et al .…”