Periodontitis is a chronic inflammatory disease associated with loss of periodontal attachment, collagen, and alveolar bone. Regeneration of periodontal tissues can be supported by the local application of enamel matrix derivative (EMD). However, periodontal regeneration remains a major and often unpredictable challenge as the result of a number of unknown factors. The authors' in vitro studies revealed that EMD stimulated the wound fill rate, proliferation, and adhesion of periodontal ligament (PDL) cells. However, in the presence of an inflammatory environment or biomechanical loading, the beneficial effects of EMD decreased significantly. EMD also stimulated the synthesis of growth factors and collagen, as well as calcium deposition, in PDL cell cultures. These beneficial effects of EMD on PDL cells were also significantly diminished by inflammation and biomechanical forces, respectively. The findings suggest that critical PDL cell functions pertinent to periodontal regeneration are reduced in an inflammatory environment and under biomechanical loading. Therefore, effective anti-infectious and anti-inflammatory periodontal treatment before the application of EMD may be critical to ensure the full regenerative capacity of the PDL tissue. Furthermore, occlusal loading of EMD-treated teeth, at least immediately following surgery, should be minimized to obtain optimal regenerative healing results. A better understanding of the interactions of growth factors and biomechanical signals will result in more powerful regenerative therapeutic strategies.