MESENCHYMAL CELL ACTIVATION IS THE RATE-LIMITING STEP OF GRANULATION-TISSUE INDUCTION

During wound repair a 3-day lag occurs between injury and granulation tissue development When full-thickness, 8-mm-round, excisional wounds were made in the paravertebral skin of outbred Yorkshire pigs and harv ested at various times, no granulation tissue was observed before day 4, Day 4 wounds were 3% filled with granulation tissue, day 5 wounds 4 8% filled, and day 7 wounds 88% filled. The prerequisites for granulat ion tissue induction are not known but hypothetically include fibrin m atrix maturation or cell activation, To examine whether matrix maturat ion was necessary, wounds were allowed to heal for 5 or 7 days and the n aggressively curetted, resulting in the formation of fresh fibrin cl ots in the newly formed wound spaces, In contrast to original wounds, mo lag phase teas observed wounds curetted on day 5 were 23% filled wi th granulation tissue 1 day later and 99% filled 3 days Inter, whereas wounds curetted on day 7 were 47% filled 1 day later and completely f illed within 2 days, Thus, granulation tissue formation resumed prompt ly and independently of fibrin clot matrix maturation, This observatio n suggested that mesenchymal cell activation might be the rate-limitin g step in granulation tissue-formation, To address this hypothesis mor e directly, cultured porcine or human fibroblasts, grown to 80% conflu ence in Dulbecco's minimal essential medium plus 10% fetal calf serum, were added to new wounds, These wounds were sealed with a freshly mad e exogenous fibrin clot, In some wounds, platelet releasate was added to the fibrin clot, Granulation tissue did not form in day 3 wounds, w hich had received either fibrin alone, fibrin and platelet releasate, or fibrin and fibroblasts. In contrast, granulation tissue was observe d in wounds receiving fibrin, human fibroblasts, and platelet releasat e, By day 4, wounds receiving cultured human fibroblasts, fibrin, and platelet releasate were 14% filled with granulation tissue compared wi th less than 4% granulation tissue in control wounds, Thus, fibroblast activation is a limiting step of granulation tissue formation, and co ntinued cell stimulation is required for accelerated development.