PROLIFERATION AND COLLAGEN SYNTHESIS OF HUMAN ANTERIOR CRUCIATE LIGAMENT CELLS IN VITRO: EFFECTS OF ASCORBATE-2-PHOSPHATE, DEXAMETHASONE AND OXYGEN TENSION

Authors


Nuffield Department of Orthopaedic Surgery, Nuffield Orthopaedic Centre, Headington, Oxford, OX3 7LD, U.K.

ABSTRACT

Clinical and experimental studies demonstrate that injured anterior cruciate ligaments (ACL) do not usually heal and that autografts used to repair the ACL rapidly weaken in the early period and take a long time to regain strength. The aim of this study was to develop an in vitro culture system in which environmental and biochemical factors influencing the proliferation and matrix synthesis of cells derived from human anterior cruciate ligaments can be studied.

Primary cultures of human ACL cells were obtained by outgrowth from explants of normal ACL obtained at knee replacement for osteoarthritis in Dulbecco's minimum essential medium (DMEM). The effects of the additives 100μm l-ascorbic acid-2-phosphate (Asc-2-P) and 10nmdexamethasone (dex) on proliferation and collagen synthesis were assessed after 4, 8 and 12 days in culture. Ligament cells were grown at 0, 5, 10 and 21% p O2in the presence of 100μmasc-2-P and 10nmdex. DNA content was assessed using the Hoechst dye method and collagen synthesis by the incorporation of 5mCi/ml [3H]proline after 3, 6 and 12 days in culture.

At 21% p O2, the presence of asc-2-P and dex induced significantly greater (P< 0.01, ANOVA) cell proliferation than with either additives alone. Greatest percentage collagen to total protein synthesis was observed when cells were grown in the presence of asc-2-P only. Least proliferation and percentage collagen to total protein synthesis was seen when both additives were omitted. Greatest cell proliferation was seen when cells were grown in 10% p O2and 5% p O2was associated with increased collagen synthesis.

These results suggest that it is possible to study the effects of environmental and biochemical factors on human ACL healing in vitro. Our data suggest oxygen can influence certain biosynthetic activities of ACL cells. Low oxygen tensions lead to an increase in collagen production by ACL cells. However early responses to injury require extensive cell proliferation which may be activated at higher p O2. Variation of p O2in ligaments during healing may therefore be an important modulator of successful repair.

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