OTA 1997 Posters - Scientific Basis for Fracture Care

Poster #70

Cytokines in Fracture Non-Union

Theodore A. Blaine, MD, Susan V. Bukata, MD, Paul T. Rubery, MD, Regis J. O'Keefe, MD, David G. Hicks, MD, M. Gordon Whitbeck, MD

University of Rochester, Rochester, New York, USA

Purpose: While a great deal of research has focused on the effect of local growth factors (BMPS, FGF, TGF-beta) on fracture healing, little attention has been given to inflammatory cytokines which may inhibit fracture healing. Cytokines (interleukins, tumor necrosis factors) produced by inflammatory cells, fibroblasts, and bone-forming cells present in the fracture callus have been shown to have potent stimulatory and inhibitory effects on these cells. In particular, interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) have been shown to stimulate osteoclastic bone resorption and inhibit bone formation in vitro. While two recent studies suggest that these inhibitory cytokines may play a role in fracture repair, the role of cytokines in fracture non-unions has not been thoroughly investigated (1,4). The present study seeks to identify and compare the presence of cytokines in normal fracture healing and in fracture non-unions.

Methods: Fracture callus from rat tibiae sustaining closed fractures with a three point bending clamp were obtained at various stages (eight days, two weeks, and four weeks) of fracture healing (3). These samples were fixed in formalin, embedded in paraffin, and tissue sections sliced and mounted on slides for histologic analysis. For the human specimens, tissue samples from clinically demonstrated fracture nonunions were collected intraoperatively for eighteen patients. These samples were prepared as described and four of these samples in addition to the rat fracture specimens were analyzed using immunohistocytochemistry for the presence of cytokines (TNF, IL-1, and IL-6). A histologic grading system was used to compare the intensity of staining for these cytokines (Table 1) (2).

Table 1. IMMUNOHISTOCHEMICAL STAINING

 Grade  Description
 0  No staining
 +  Weakly positive focal staining.
 ++  Strongly positive focal staining.
 +++  Strongly positive diffuse staining.
 ++++  Intensely positive staining in all cells.

Results: In rat tibiae undergoing normal fracture repair, low levels of TNF-alpha and interleukin-1 were demonstrated at all time points (8 days, 2 weeks, and 4 weeks post-fracture) (Table 2). Fracture callus was also analyzed for the presence of bFGF, which is known to be demonstrated at high levels in normal fracture callus. All specimens demonstrated high levels of bFGF, indicating a normal fracture healing response.

Table 2. RAT FRACTURE UNIONS

 Tissue Sample  Histology

 Immunohistochemistry

 TNF  IL-1  bFGF
 Rat Fracture (8 days)  Fibrocartilage  +  0  +++
 Rat Fracture (2 weeks)  Endochondral bone  0  +  ++++
 Rat Fracture (4 weeks)  Endochondral bone  0  +  +++

Unlike the normally healing rat fracture callus, tissue sections from human fibrous non-unions stained strongly positive for the presence of TNF-alpha, IL-1, and IL-6, compared to the weak staining in normal fracture callus (Table 3).

Table 3. HUMAN FRACTURE NON-UNIONS

 Tissue Sample  Histology

 Immunohistochemistry

 TNF  IL-1  IL-6
 Subject 1  Fibrous tissue  +++  +++  +++
 Subject 2  Fibrous tissue  ++  ++  ++
 Subject 3  Fibrocartilage  ++++  +++  ++++
 Subject 4  Fibrocartilage  ++++  +++  ++++

Discussion: These findings suggest that cytokines (TNF-alpha and IL-1) which are normally present at low levels in fracture healing may be present at high levels in fracture non-unions. These cytokines are known to stimulate bone resorption and prevent bone formation, and therefore may play a causative role in the abnormal fracture healing response leading to non-union. While a direct correlation cannot be made from these findings due to species variation and insignificant numbers of specimens, further studies are indicated to demonstrate the significance of these observations.

1. Einhorn, T.A.; Majeska, R.J.; Rush, E.B.; Levine, P.M.; and

Horowitz, M.C.: The Expression of Cytokine Activity by Fracture Callus.

J. Bone and Mineral Res., 10(8): 1272-81, 1995.

2. Hicks, D.G., Judkins, A.R., Sickel, J.Z., Rosier, R.N., Puzas, J.E., and O'Keefe, R.J. Granular Histiocytosis of Pelvic Lymph Nodes following Total Hip Arthroplasty. J. Bone and Joint Surg. 78-A, 4:482-96, April, 1996.

3. Hughes, S.S., Hicks, D.C., O'Keefe, R.J., Hurwitz, S.R., Crabb, I.D., Krasinskas, A.M., Loveys, L., Puzas, J.E., and Rosier, R.N. Shared

Phenotypic Expression of Osteoblasts and Chondrocytes In Fracture Callus.

J. Bone and Min. Res., 10:4, 533-44, 1995.

4. Milgram, J.W.: Non-union and Pseudarthrosis of Fracture Healing: A

Histopathologic Study of Ninety-five Human Specimens. Clin. Orth. Rel.

Res., 268: 203-13, 1991.