Arch Orthop Trauma Surg (1990) 109: 268-271

Ach"°fOrthopaedic ..dTrauma Surgery © Springer-Verlag1990

The polymorphonuclear leukocyte: has it a role in fracture healing? B. Grogaard 1, B. Gerdin 2, and O. Reikerfis 1 1Department of Surgery, Orthopedic Section, Ullev~l University Hospital, Oslo, Norway 2Department of Surgery, University Hospital, Uppsala, Sweden

Summary. The aim of the present study was to assess whether the presence of p o l y m o r p h o n u c l e a r leukocytes in the first stage of fracture repair was of any importance. In anesthetized male Wistar rats, a transverse o s t e o t o m y was p e r f o r m e d at midshaft in one femur and immediately stabilized by an intramedullary Kirschner nail. The animals were allowed unprotected weight bearing immediately. Nine animals were m a d e neutropenic before operation by injection of a specific rat p o l y m o r p h o n u c l e a r leukocyte antiserum (antineutrophil serum, ANS) raised in sheep. The n u m b e r of circulating polymorphonuclear leukocytes were kept below 20% of their normal value for 72 h postoperatively by intraperitoneal injections of ANS every 12 h. Control animals were injected with the same amount of normal sheep serum (NSS). All animals received cefuroxime (100 mg/kg day) concomitantly with the ANS injections. Six weeks after operation the animals were killed, the amount of callus formation was measured, and the bones were radiologically examined. The nails were then r e m o v e d and b o t h the healing fractures and the n o n o p e r a t e d femurs mechanically tested for bending. T h e r e were no differences in the amount of callus m e a s u r e d or in radiological healing. H o w e v e r , there was a significantly higher bending moment in femurs from animals treated with ANS than in those given NSS (P < 0.02). No differences were observed in rigidity or total energy absorption.

The first stage of fracture repair is characterized by extravasation of blood followed by aseptic inflammation. The h e m a t o m a around the fracture site is invaded by a variety of blood elements, including polymorphonuclear leukocytes. For years it has generally been accepted that the inflammatory response is beneficial and indeed necessary in all sorts of tissue repair. Recent data, howOffprint requests to." B. GrCgaard, Department of Surgery, Orthopedic Section, Ullevfil Hospital, Kirkeveien 166, N-407 Oslo 4, Norway

ever, indicate that the invasion of polymorphonuclear leukocytes into damaged tissues, as in myocardial infarction [13], lung injury [17], skeletal muscle ischemia [7], and the repairing process after intestinal anastomosis [5] m a y aggravate the tissue damage and jeopardize tissue repair (for survey see [4]). Recently, therefore, interest has been focused m o r e on the possible side effects than on the beneficial role of polymorphonuclear leukocytes in host defense. The aim of the present study was to asses whether the presence of these leukocytes in the first stage of fracture repair was of any significance.

Materials and methods A n i m a l preparation Male Wistar rats (250-275g, M¢llegaard, Copenhagen) were used. Following anesthesia induced with Dormicum (midazolam 1 mg/ml, Roche) and Hypnorm (fluanisone 10 mg/ml, fentanyl citrate 0.315 mg/ml, Roche) in equal amounts, given intramuscularly, 0.5 ml/100 g b.w., the left femur was exposed through a lateral incision. A transverse osteotomy was made at the midshafl of the femur with a fine-toothed circular saw blade mounted on an electric drill. The osteotomy was immediately stabilized by a 1.2-mm intramedullary Kirschner nail. The wounds were then closed in two layers. The animals tolerated the operation well, and unprotected weight bearing was allowed immediately. Six weeks after the operation the animals were killed and the femurs carefully dissected. The frontal and transverse diameter of the callus mass was measured by a sliding caliper, and the quantity of the callus was expressed as the cross-sectional area, assuming it to be an ellipse. The bones were then radiologically examined and the fractures classified as healed, nearly healed, or not healed according to the presence of bridging callus (Fig. 1) [12]. The nails were then removed and both fractured and nonfractured femurs were mechanically tested for bending as described by Engs~eter et al. [2]. A standard hydraulic testing machine was run at a constant rate (0.04 rad/s). The load values were transferred to a chart recorder displaying the load-deformation curve. The strength of the bones was calculated as the bending moment necessary to produce fracture. The bending rigidity was determined from the slope of the linear elastic part of the curve.

B. GrCgaard et al.: Polymorphonuclear leukocytes in fracture healing


Preparation of antineutrophil serum

Experimental groups

Antineutrophil serum (ANS) was prepared as described by Sandler et al. [15], except that the immunization took place in sheep. Rat polymorphonuclear leukocytes (50 x 106, 0.5 ml) were mixed with an equal volume of Freund's complete adjuvant and injected intramuscularly every 2nd week into sheep. Blood was collected and the antisermn then processed as described previously [15]. Normal sheep serum (NSS) for control experiments was collected prior to immunization and treated in the same way as the ANS.

Group 1 (n = 9). In this group the animals were injected with 3 ml ANS intraperitoneaUy 10 h before operation. Another 2 ml was administered immediately postoperatively, after which l ml was given every 12 h up to 72 h postoperatively. Prior to the first injection, at operation, and daily thereafter, blood samples were drawn from a superficial vein in the right hindlimb for leukocyte, monocyte, and platelet counts. To avoid infections, cefuroxim (100 mg/ kg b.w. per day, Glaxo) was given intraperitoneally concommitantly with the serum injections. Group 2 (n = 8). The animals in this group were treated with NSS and cefuroxim according to the protocol described for group 1.

Statistical evaluation The Wilcoxon-White two-sample ranks test was used. The results are expressed as mean ± SD. Changes are considered significant when P < 0.05.


Fig. 1. Radiological examination of the femurs 6 weeks after the fracture. From left to right: not healed, nearly healed, and healed

T r e a t m e n t with A N S ( G r o u p 1) r e s u l t e d in d e p l e t i o n o f p o l y m o r p h o n u c l e a r l e u k o c y t e s to less t h a n 20% of t h e b a s e l i n e v a l u e ( T a b l e 1). T h e a n i m a l s w e r e n e u t r o p e n i c as l o n g as t h e injections w e r e given, i.e. for 72 h p o s t o p e r a t i v e l y . B y 9 6 h a f t e r o p e r a t i o n all a n i m a l s h a d n o r m a l i z e d p o l y m o r p h o n u c l e a r l e u k o c y t e counts. N o significant c h a n g e s o r d i f f e r e n c e s w e r e o b s e r v e d in the n u m b e r o f m o n o n u c l e a r cells d u r i n g t h e o b s e r v a t i o n p e r i o d ( T a b l e 1). T h e n u m b e r o f p l a t e l e t s s h o w e d a slight d e c r e a s e in b o t h g r o u p s d u r i n g t h e first 3 days after o p e r a tion ( T a b l e 1). I n a n i m a l s t r e a t e d with NSS t h e n u m b e r o f p l a t e l e t s n o r m a l i z e d t h e r e a f t e r , w h e r e a s in A N S - t r e a t e d a n i m a l s an i n c r e a s e to 50% a b o v e b a s e l i n e was o b served.

Table 1. Number of polymorphonuclear leukocytes, mononuclear cells, and platelets in the blood in animals given antineutrophil serum or normal sheep serum. Numbers in parentheses are % of baseline - 10 h


24 h

48 h

72 h

96 h

120 h

0.9±0.2 (100%)

0.2±0.4 (17 ± 36)

0.1+0.2 (11 ± 20)

0.1+0.1 (17 ± 16)

0.1+0.2 (17 + 18)

1.310.7 (122 ± 96)

1.8+1.2 (208 ± 162)

0.8_+1.3 (87 ± 37)

0.8 ± 0.2 (100%)

0.8 + 0.3 (99 + 28)

1.5 ± 0.4 (203 ± 83)

0.9 ± 0.1 (132 ± 20)

0.8 ± 0.3 (118 ± 64)

0.9 ± 0.5 (133 ± 83)

0.9 _+0.6 (132 ± 108)

1.2 ± 0.7 (139 +_65)

Mononuclear cells ANS 9.2 + 0.8 (100%)

6.9 ± 2.2 (74 + 20)

7.6 ± 1.5 (81 ± 19)

6.3 + 1.1 (69 -+ 13)

6.5 + 1.6 (71 + 17)

10.9 ± 1.7 (119 ± 18)

13.0 _+2.6 (143 ± 31)

9.0 ± 2.2 (96 ± 21)

8.5+1.6 (100%)

8.5+1.5 (98 _+24)

7.3_+1.8 (90 ± 33)

7.5+1.4 (88 + 24)

7.7+1.7 (93 ± 12)

8.4+1.1 (102 ± 23)

9.7±1.4 (121 + 37)

8.5+1.9 (107 ± 46)

373 +_71 (100%)

225 + 78 (61 ± 21)

238 ± 94 (64 ± 26)

244 ± 150 (67 + 41)

nd -

506 ± 129 (137 ± 26)

591 + 59 (164 k 37)


340 + 97 (100%)

279 ± 36 (82 + 11)

262 ± 89 (77 _+26)

nd -

282 ± 60 (82 + 18)

318 ± 67 (93 ± 20)

362 ± 94 (105 ± 27)




NSS Platelets ANS


PMNL, polymorphonuclear leukocytes; ANS, antineutrophil serum; NSS, normal sheep serum; nd, not determined

6 weeks

B. GrCgaard et al.: Polymorphonuclearleukocytesin fracture healing

270 x

10-' Nm

Nm x dg

10 -~ N m / d




Fig. 2. a Bendingmoment, b rigidity,and c total energyabsorption at the fracture site in animals given antineutorphil serum (ANS) (N) or normal sheep serum (NSS) ([3). Intact femurs to the left

Radiological examination revealed that the fractures healed by formation of external callus (Fig. 1). There was no significant difference in radiological healing or measured callus masses. In ANS-treated animals the bending moment at the fracture site was significantly higher than in NSS-treated animals (Fig. 2a). No significant differences were observed concerning rigidity or total energy absorption (Fig. 2b, c).


From these results it seems that if polymorphonuclear leukocytes have any influence on fracture healing, their effects are negative: that is, a transverse femoral midshaft osteotomy had a significantly higher bending moment 6 weeks after operation in animals made neutropenic prior to the operation. In this study experimental fractures in rats were pinned, but not rigidly, so they healed by the production of external callus. It is well known that fracture healing under unstable conditions take place by the formation of external bridging callus. With increasing maturity of the callus tissue, the fracture proceeds to consolidation. The aim of fracture treatment is recovery of the mechanical properties of the bone. In this study the bones were tested in bending, and we found that they gained strength faster in animals deprived of their polymorphonuclear leukocytes. Furthermore, although not significant, there was a tendency to increased rigidity at the fracture site in these animals. The values in total energy absorption were quite similar in the two groups. However, a fracture showing early maturity usually exhibits high stiffness and thereby relatively reduced energy absorption. The presence or absence of union was evaluated by radiographic examinations. It has been shown that this can be done reliably [12]. According to both radiographic evaluation and the measurements of callus production, there were no significant differences between the two groups. Although the results of our study imply that neutropenia attenuates fracture healing, the mechanism re-

mains obscure. Theoretically, polymorphonuclear leukocytes could release at least three groups of products that could negatively interfere with fracture repair. 1. Polymorphonuclear leukocyte granules contain a variety of substances, including elastase, collagenase, cathepsins, lysozymes, cationic proteins, lactoferrin, and myeloperoxidase [9, 11, 16]. 2. Activated polymorphonuclear leukocytes possess a nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase on their plasma membrane that reduces molecular oxygen to the superoxide anion free radical (O½-), with concomitant oxidation of cytosolic NADPH [1]. This induces the production of a variety of oxidizing agents such as hydrogen peroxide (H202), the hydroxyl free radical (HO) and hypochlorous acid (HOC1). This is an important mechanism in phagocytes for killing bacteria and also takes place in inflammatory reactions at the fracture site. Oxygen-derived free radicals are thereby released in the extracellular compartment which is poorly protected by endogenous scavengers [3, 18]. 3. Upon stimulation of neutrophils, phospholipase A2 is activated and acts to release arachidonic acid from the cells' phospholipids. This free arachidonic acid may serve as a substrate for both the cyclooxygenase and lipoxygenase pathways [14]. There seems little doubt, then, that several of these factors could have negative effects on fracture repair. Parallel to the direct cellular toxicity, the inflammatory response due to the polymorphonuclear leukocytes might aggravate the already existing ischemia [10], e.g., phagocyte-generated superoxide is known to increase capillary permeability [6, 8], leading to edema and increased interstitial pressure. As the interstitial pressure approaches intravascular pressure, local circulatory shutdown occurs, producing ischemia, References

1. Babior BM (1978) Oxygen-dependent microbial killing by phagocytes. N Engl J Med 298 : 659-668,721-725 2. Engsmter LB, Ekeland A, Langeland N (1978) Methods for testing the mechanicalproperties of the rat femur. Acta Orthop Scand 49 :512-518 3. Freeman BA, Crapo JD (1982) Free radicals and tissue injury. Lab Inv 47 : 412-426 4. Harlan JM (1985) Leukocyte-endothelialinteractions. Blood 65 : 513-525

B. Gr~agaard et al.: Polymorphonuclear leukocytes in fracture healing 5. HCgstmm H, Haglund U (1985) Neutropenia prevents decrease in strength of rat intestinal anastomosis; partial effect of oxygen free radical scavengers and allopurinol. Surgery 99: 716-720 6. Korthuis RJ, Granger DN, Townsley MI, Taylor A E (1985) The role of oxygen-derived free radicals in ischemia-induced increases in canine skeletal muscle vascular permeability. Circ Res 57 : 599-609 7. Korthuis RJ, Grisham MB, Granger DN (1988) Leukocyte depletion attenuates vascular injury in postischemic skeletal muscle. Am J Physiol 254 : H823-827 8. Ley K, Arfors K-E (1982) Changes in macromolecular permeability by intravascular generation of oxygen-derived free radicals. Microvasc Res 24 : 25-33 9. Macartney HW, Tschesche H (1983) Latent and active human polymorphonuclear leukocyte collagenase: isolation, purification and characterization. Eur J Biochem 130: 71-78 10. McCord JM (1987) Oxygen-derived radicals: a link between reperfusion injury and inflammation. Fed Proc 42:2402-2406 11. Murphy KR (1976) The neutrophile. Plenum, New York 12. M¢lster AO, Gjerde NR (1984) Biomechanical effects of instability on fracture healing in the rat. Acta Orthop Scand 55: 342-346

271 13. Romson JL, Hook BG, Kunkel SL, Abrams GD, Schork MA, Lucchesi BR (1983) Reduction of the extent of ischemic myocarnal injury by neutrophil depletion in the dog. Circulation 67:1016-1023 14. Samuelsson B, Hammarstr6m S, Borgeat P (1979) Pathways of arachidonic acid metabolism. In: Weissman G, Samuelsson B, Paoletti R (eds). Raven, New York, p 405 15. Sandier H, H6gstorp H, Lundberg C, Gerdin B (1987) Antiserum-induced neutropenia in the rat: characterization of a rabbit anti-rat neutrophil serum. Br J Pathol 68 : 71-80 16. Starkey PM, Barrett AJ, Burleigh MC (1977) The degradation of articular collagen by neutrophil proteinases. Biochem Biophys Res Corn 483 : 386 17. Till GO, Johnson KJ, Kunkel R, Ward PA (1982) Intravascular activation of complement and acute lung injury. Dependency on neutrophils and toxic oxygen metabolites. J Clin Invest 69:1126-1135 18. Weiss SJ (1986) Oxygen, ischemia, and inflammation. Acta Physiol Scand 126 [Suppl 548] : 9-38

Received December 27, 1989

The polymorphonuclear leukocyte: has it a role in fracture healing?

The aim of the present study was to assess whether the presence of polymorphonuclear leukocytes in the first stage of fracture repair was of any impor...
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