Serum complement system activation in normal healing and atrophic non-union of human long bone fractures

The complement system has an important role in physiological bone healing, as studied mainly in animal models and local bone tissues. However, the role of the complement system in human fractures, particularly at the systemic level, remains insufficiently characterized. This study aimed to investigate the activation levels of the serum complement system during three healing phases (inflammation, repair and remodeling) of normal healing of long bone fractures and in patients diagnosed with fracture non-union.

Blood samples were obtained from two groups of long bone fracture patients (normal healers and non-union) and healthy controls with no fractures. Blood samples from patients with normally healed fractures were collected at 1 week, 1 month, and 4-6 months post-fracture. Blood samples from patients diagnosed with non-union were collected at 1-year post-fracture. The serum samples were processed using mass spectrometry to quantify the complement protein expression. The ELISA was used for validation. Ingenuity Pathway Analysis (IPA) software was used for molecule-pathway interactions.

The classical complement pathway components, complement C1s and C1r, were significantly increased during the inflammation phase of normally healed fractures but reduced subsequently. The levels of serum C3, C3a, and C9 were significantly greater in the inflammatory phase than in the other phases. No significant differences were observed for other complement pathway components, complement factor B (CFB), complement factor H and I (CFH, CFI), ficolin 2 and 3 (FCN2, FCN3) and mannan-associated serine protease-1 (MASP1) when comparing the three phases. In fracture non-union, the serum MASP1 level was significantly higher than that of normal fracture healers and healthy controls. The IPA analysis showed a link between MASP1 as a part of the lectin pathway and damage in bone and cartilage.

Collectively, our data indicate temporal changes of the serum complement system with activation via the classical pathway, particularly during the inflammatory phase of normal healing of human bone fractures. Furthermore, systemic MASP1 levels were high in non-united fractures, indicating that the lectin pathway plays a unique role in abnormal fracture healing. This data will offer fresh avenues for utilizing complement system components, such as follow-up biomarkers and therapeutic targets, in bone injuries and diseases.