Systemic inflammation and fracture healingOkan Bastian,* Janesh Pillay,† Jacqueline Alblas,‡ Luke Leenen,* Leo Koenderman,†

and Taco Blokhuis*,1

Departments of *Surgery, †Pulmonary Diseases, and ‡Orthopedics, University Medical Center Utrecht, The Netherlands

RECEIVED AUGUST 9, 2010; REVISED NOVEMBER 12, 2010; ACCEPTED DECEMBER 10, 2010. DOI: 10.1189/jlb.0810446

ABSTRACTApart from their pivotal role in the host defense againstpathogens, leukocytes are also essential for bone re-pair, as fracture healing is initiated and directed by aphysiological inflammatory response. Leukocytes infil-trate the fracture hematoma and produce severalgrowth and differentiation factors that regulate essen-tial downstream processes of fracture healing. Sys-temic inflammation alters the numbers and propertiesof circulating leukocytes, and we hypothesize thatthese changes are maintained in tissue leukocytes andwill lead to impairment of fracture healing after majortrauma. The underlying mechanisms will be discussedin this review. J. Leukoc. Biol. 89: 669–673; 2011.

IntroductionFractures in severely injured patients require a significantlylonger healing time compared with isolated fractures and carryan increased risk of developing nonunion [1], which is failureof the bone healing process to unite the bone fragmentswithin 9 months after injury [2]. This impairment of fracturehealing not only has a detrimental effect on quality of life ofthe patient but also carries a substantial cost to society. Thedirect costs of treating nonunions have been estimated be-tween $31.144 and $34.413 per nonunion in the United King-dom with considerable additional costs as a result of the lossof productivity of patients during the period of postinjury dis-ability [3].

Several local changes after major trauma have been well rec-ognized as factors that contribute to impairment of fracturehealing, such as poor condition of the surrounding soft tissueand open fracture sites [1]. Systemic changes after majortrauma, however, have received far less attention as possiblerisk factors that contribute to impairment of fracture healing.Systemic inflammation, which is characterized by leukocytosis,priming of leukocytes, and systemic release of cytokines, is typ-ically induced in trauma patients within 24 h after admission[4]. These patients are also at risk for prolonged systemic in-flammation, as surgical intensive care unit patients maintain a

systemic inflammatory response during their unit stay [5]. Fac-tors that contribute to prolonged systemic inflammation aftermajor trauma include the high incidence of sepsis after majortrauma [6] and extended trauma by multiple, additional surgi-cal interventions [7].

NORMAL FRACTURE HEALING

Bone tissue can heal through direct (primary) cortical healing orindirect (secondary) healing by formation of a callus, which isthe newly developed mass of fibrocartilaginous and osseous tissuesurrounding the fracture site. Primary bone healing is rare anddoes not involve major influx of inflammatory cells into the frac-ture hematoma [8]. As a result of the absence of a major inflam-matory component, primary bone healing is less likely to be af-fected by the cellular changes that are caused by systemic inflam-mation and therefore, will not be discussed in this review.

In most fractures, a certain amount of mechanical instabilityleads to interfragmentary movement and thus, to secondary frac-ture healing. This involves a local inflammatory reaction followedby a proliferative and remodeling phase that leads to reconstruc-tion of bone. The inflammatory phase is initiated when disrup-tion of the vasculature leads to a hematoma around the fracturesite that becomes infiltrated by inflammatory cells.

The concentration of leukocytes within the fracture hematomaremains similar to their concentration in the peripheral circula-tion for several hours after injury [9]. Within 24 h, however,there is a major influx of neutrophils into the fracture hema-toma, making neutrophils the predominate leukocyte within thefracture hematoma [10]. Hereafter, they become replaced bymacrophages [11]. Recruited neutrophils are thought to mediatethis switch by producing several macrophage chemoattractants,such as MCP-1, also known as CCL2, and IL-6 [12, 13]. After thismacrophage phase, T-lymphocytes are selectively recruited intothe fracture hematoma and subsequent granulation tissue,whereas practically no B-lymphocytes are found at any stage offracture healing [11]. An extended overview of the current litera-ture that addresses the composition and potential of the earlyfracture hematoma has been reviewed recently by Kolar and col-leagues [14].

1. Correspondence: Department of Surgery, UMC Utrecht, Heidelberglaan100, HP G04. 228, 3508 GA Utrecht, The Netherlands. E-mail: t.j.blokhuis@umcutrecht.nl

Abbreviations: –/–�knockout, BMP�bone morphogenetic protein,MSC�multipotent stromal cell, SOX�sex-determining region y-relatedhigh-mobility group box

Mini-Review

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Within several days, the fracture hematoma becomes intrinsi-cally osteogenic and angiogenic, which is demonstrated by thefact that transplantation of the 4-day-old fracture hematoma intomuscle tissue induces extraskeletal bone formation [15] and an-giogenesis [16]. The composition of the fracture hematoma isestablished predominantly by factors that are produced by infil-trated inflammatory cells, illustrating the importance of local con-trolled inflammation for adequate bone repair. Disturbance of theinflammatory phase of bone repair is known to impair fracture heal-ing, as removal of the fracture hematoma at 30 min or 2 or 4 daysafter injury in a rat femur fracture model leads to a significant de-crease in bone mechanical characteristics [17]. Moreover, irrigationand debridement of the fracture hematoma on the first and secondday after osteotomy in a rabbit model consistently lead to atrophicfracture nonunion [18].

Studies that have compared early mRNA expression of genesthat regulate cartilage, bone, and vessel formation betweenstandard bone healing and mechanically induced, delayedbone healing in sheep found that at Day 7 after osteotomy,expression of Sox-9, VEGFR2, and tyrosine kinase with Ig-likeand EGF-like domain 2 was significantly higher in the delayedhealing group, and expression of TGF-�1 and VEGF was signif-icantly lower compared with the standard healing group [19,20]. This suggests that the composition of the fracture hema-toma, within days after injury, correlates with the eventual out-come of the bone healing process.

The inflammatory phase is followed by the proliferativephase, which involves a combination of intramembranous andendochondral ossification. During intramembranous ossifica-tion, bone tissue is formed directly by committed osteopro-genitor cells that reside in the periosteum without first form-ing cartilage. Endochondral ossification, in contrast, involvesthe recruitment, proliferation, and differentiation of undiffer-entiated MSCs into chondroblasts that form cartilage, whicheventually becomes replaced by bone [21]. The initially avascu-lar cartilage provides mechanical support to the fracture and isconverted to bone when hypertrophy of chondrocytes pro-gresses, which causes calcification of cartilage matrix and con-current invasion of vasculature [22]. As vasculature begins toinvade, the calcifying hypertrophic chondrocytes are removedby chondroclasts, after which, undifferentiated MSCs that dif-ferentiate into osteogenic cells form woven bone.

It remains unclear which cells play an essential role in direct-ing MSCs toward the osteogenic lineage in vivo. However, it hasbeen shown that macrophages [23] and activated T-lymphocytes[24] stimulate osteogenic differentiation of MSCs in vitro.

Osteogenic differentiation of MSCs is stimulated by severalfactors of which BMPs are the most powerful known [25].BMPs are members of the TGF-� family that bind to type Iand type II serine-threonine kinase-containing receptors [26].Binding of these receptors leads to phosphorylation of specificdownstream effector proteins called Smads [27].

Inflammatory cytokines also have the potential to stimulateosteogenic differentiation of MSCs. A mixture of TNF-�,TGF-�, IFN-�, and IL-17 stimulates osteogenic differentiationof MSCs in vitro [24], whereas individually or in combinationsof up to three, these inflammatory cytokines fail to stimulateosteogenic differentiation. These data imply that osteogenic

differentiation of MSCs is modulated by a complex ratio ofcytokines and that alteration of inflammatory cytokine concen-trations can affect differentiation of MSCs.

The final stage of fracture repair encompasses the remodel-ing of the woven bone into the original laminar bone configu-ration by osteoclasts that first create erosive pits on the bonesurface, on which new bone is then laid down by osteoblasts.This process eventually restores the laminar configuration andstrength of the original bone tissue [28].

In summary, the hypothesis that inflammation is essential forfracture healing is supported by the findings that removal of theearly fracture hematoma leads to a significant decrease in bonemechanical characteristics [17], and repetitive irrigation of the earlyfracture hematoma leads to atrophic fracture nonunion [18].

SYSTEMIC INFLAMMATION ANDFRACTURE HEALING

Although local, controlled inflammation seems essential forbone repair [15, 18], previous studies have shown that in-creased or prolonged inflammation, within the fracture hema-toma, impairs fracture healing. For instance, local applicationof semisoluble aminated glucan at the fracture site, whichleads to release of inflammatory cytokines by local inflamma-tory cells, has been shown to result in immature hypertrophiccallus formation with reduced bone mechanical properties[29]. Other studies have shown that fractures that are accom-panied by severe overlying muscle crush injury in rats demon-strate delayed healing, which is consistent with a detrimentaleffect of local hyperinflammation on fracture healing [30].

Systemic inflammation, induced by i.p. injection of LPS intorats with femur fractures, has shown to induce formation of ahypertrophic and immature callus with reduced bone mechanicalcharacteristics [31]. However, the mechanism through which sys-temic inflammation affects the local inflammatory phase of bonerepair remains to be addressed adequately.

To speculate on a mechanism through which systemic inflam-mation impairs bone healing, it is relevant to first address the in-flammatory changes that occur during systemic inflammation.

Tissue injury leads to the release of endogenous damage-associ-ated molecular patterns that activate innate immune cells [32].Activation of the immune system is associated with an increasedheart rate and respiratory rate and an increased or decreasedbody temperature, and leukocyte count [4]. The presence of atleast two of these symptoms is termed systemic inflammatory re-sponse syndrome, and the severity of systemic inflammation posi-tively correlates with the extent of injury [33].

The increased leukocyte count is established predominantlyby the rapid release of neutrophils from the bone marrow intothe peripheral circulation [34, 35], which leads to a function-ally heterogeneous neutrophil compartment that consists ofyoung, banded neutrophils with a refractory phenotype andsegmented neutrophils with a primed phenotype [34]. Neutro-phils form the first natural immunological defense againstpathogens and are involved in debridement of injured tissue.These cells are relatively short-lived and circulate for severaldays [36]. Their concentration increases rapidly in the periph-eral circulation during systemic inflammation and peaks within

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several hours [34] until 3 days after injury [35]. Neutrophilsbecome preactivated by several factors that are up-regulatedafter severe trauma [37], and the neutrophil pool can remainprimed until 13 days after trauma [38]. These priming factorsinclude TNF-�, IL-8, and LPS [35]. Primed neutrophils areprone to respond to inflammatory stimuli. The importance ofthis process is illustrated by the fact that neutrophils isolatedfrom trauma patients show a primed phenotype characterizedby enhanced chemotaxis toward IL-8 in vitro [39]. Moreover,it has been shown that systemic administration of �-glucan inrats, which induces a primed state of neutrophils [40], resultsin a more than 150% increase of neutrophil infiltration intos.c.-implanted polyvinyl alcohol sponges [41]. In addition, sys-temic �-glucan treatment resulted in a more than 150% in-crease in neutrophil number in the BAL fluid of Escherichia colipneumonic animals [41], which also illustrates the enhancedmigratory function of primed neutrophils.

It is, therefore, tempting to speculate that neutrophilia andneutrophil priming, leading to an increased influx of neutro-phils into the fracture hematoma, play an important role inthe pathogenesis of impaired fracture healing after systemicinflammation (Fig. 1). This is based on the finding that sys-temic neutrophil reduction with antineutrophil serum in ratswith femur fractures induces enhanced fracture healing [42].Moreover, systemic neutrophil reduction in rats with growthplate injury leads to a 60% reduction of neutrophils within thefracture hematoma and also improved bone repair [10]. Ratstreated with antineutrophil serum showed decreased cartilagi-nous tissue, chondrogenic transcription factor Sox-9, and carti-lage matrix collagen II, along with an increased bone matrixprotein osteocalcin and osteogenic transcription factor core-binding factor �-1 [10]. This implies that neutrophils maystimulate chondrogenesis and inhibit osteogenesis. An in-creased or prolonged influx of neutrophils into the fracturehematoma during systemic inflammation may therefore impairfracture healing through overstimulation of chondrogenesisand inhibition of osteogenesis.

In addition to this hypothesis, several other mechanismsthrough which systemic inflammation may impair fracturehealing should be considered.

It is known, for instance, that monocytes can differentiate to-ward several subtypes of macrophages depending on environ-mental circumstances. IFN-� and LPS exposure induces a “classi-cally activated”, proinflammatory phenotype of macrophages, andIL-4 or IL-13 induces an “alternatively activated”, regenerativephenotype [43]. It has been shown that monocyte subsets oftrauma patients exhibit preferential differentiation toward proin-flammatory phenotypes [44]. The finding that macrophages,which were exposed to LPS, lose their ability to stimulate osteo-genic differentiation of MSCs in vitro [23] raises the question ofwhether altered differentiation of monocytes during systemic in-flammation has a negative effect on fracture healing.

It remains unknown, however, whether macrophages withinthe fracture hematoma also exhibit enhanced differentiationtoward a proinflammatory phenotype after systemic inflamma-tion and whether this has a significant effect on bone repair.

In addition, it has been shown that severe injury induces unre-sponsiveness of T-lymphocytes, characterized by simultaneous,diminished proliferation and IL-2 production [45, 46]. The find-ing that unactivated T-lymphocytes fail to stimulate osteogenicdifferentiation of MSCs in vitro, while activation of T-lymphocytesleads to stimulation of osteogenic differentiation [24], impliesthat unresponsiveness of T-lymphocytes during systemic inflam-mation may also be involved in reduced osteogenesis after sys-temic inflammation. The assumption that T-lymphocyte activationis essential for fracture healing, however, is contradicted by a re-cent study [47], which showed accelerated fracture healing inRAG-1�/� mice that lack functional B and T lymphocytes [48].Although this study implies a negative effect of the adaptive im-mune system on fracture healing, it is difficult to determinewhich lymphocyte subsets predominantly mediate this negativeeffect on bone repair. Absence of the RAG-1 gene, for instance,may predominantly improve fracture healing by disturbing thefunction of � /� T-lymphocytes. This assumption is supported bythe finding that � TCR–/– mice, which lack �/� T-lymphocytes,

Figure 1. Hypothetical model of the early inflammatoryphase of fracture healing during systemic inflammation.Neutrophilia and neutrophil priming during systemic in-flammation lead to an increased influx of neutrophils intothe fracture hematoma, which alters the local concentra-tions of growth and differentiation factors and thereby,disturbs downstream processes of bone repair.

Bastian et al. The effect of systemic inflammation on fracture healing

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also exhibit improved fracture healing [49]. In addition, it is diffi-cult to assess whether bone repair is improved predominantly bydisturbance of the regulatory or effector function of �/� T cells.It has been shown that �/� T cells play an important role in therecruitment of inflammatory cells toward inflammatory sites,which is illustrated by the fact that �/� T-lymphocyte-deficientmice showed a sixfold reduction in cellular infiltrate into polyvinylalcohol sponges that were s.c.-implanted beneath sites of thermalinjury compared with WT mice [50]. Several studies also suggest animportant role of �/� T-lymphocytes in neutrophil-mediated tissuedamage after systemic inflammation. This is illustrated by the factthat WT mice subjected to thermal injury show neutrophil accumu-lation in the lung and small intestines, and �/� T-lymphocyte-defi-cient mice did not show a similar increase in neutrophil tissue con-tent [51]. It is therefore possible that improved fracture healing inRAG-1–/– mice and � TCR–/– mice is mediated by a decreased influxof neutrophils into the fracture hematoma. The finding that �/� Tcells exhibit increased expression of activation markers during sys-temic inflammation [52] additionally raises the question of whetherincreased activity of �/� T cells during systemic inflammation in-duces an increased influx of neutrophils into the fracture hematomaand therefore, impairs fracture healing.

In summary, systemic inflammation induces several changesin leukocyte characteristics, such as neutrophil priming [37,38], altered monocyte differentiation [44], T-lymphocyte unre-sponsiveness [45, 46], and increased �/� T cell activation [52].The effect of these changes on fracture healing has not beeninvestigated, and therefore, it is difficult to determine whichmechanism mediates impairment of fracture healing after sys-temic inflammation.

It is tempting, however, to speculate that neutrophilia andneutrophil priming during systemic inflammation induce anincreased influx of primed neutrophils into the fracture hema-toma, which leads to an aberrant concentration of growth anddifferentiation factors and therefore, disturbs downstream pro-cesses of fracture healing (Fig. 1). This hypothesis is supportedby the findings that primed neutrophils exhibit enhanced mi-gration toward inflammatory sites in vivo [41], and systemicneutrophil reduction improves fracture healing [42], stimu-lates osteogenesis, and inhibits chondrogenesis [10].

ANGIOGENESIS-OSTEOGENESIS COUPLING

Disruption of the inflammatory phase of fracture healing maynot only affect osteogenesis but also angiogenesis, as these twoprocesses are linked [53]. Whereas cartilage is an avascularand hypoxic mesenchymal tissue, bone is highly vascularized[54], which suggests that angiogenesis is essential for endo-chondral ossification. It has been shown that osteoblasts canbe stimulated to secrete VEGF in culture by several BMPs [55].The action of BMPs on osteoblasts establishes a positive-feed-back loop, where the BMP-induced VEGF release causes vesselin-growth, leading to the delivery of osteogenic precursor cellson which BMPs will act to further increase VEGF concentra-tions at the fracture site [56].

Apart from osteoblasts, macrophages [57] and neutrophils[58] also produce VEGF. Release of VEGF by neutrophils canbe stimulated by PMA, fMLP, and TNF-� [58]. It has been

shown that VEGF not only regulates recruitment, survival, andactivity of endothelial cells, osteoblasts, and osteoclasts but alsoregulates cartilage maturation and resorption [59, 60]. As os-teogenesis and angiogenesis are linked, failure of leukocytes toadequately stimulate osteogenesis after systemic inflammationmay therefore further impair fracture healing by also affectingangiogenesis. The fact that proinflammatory macrophages failto stimulate autologous BMP production of MSCs [23] maytherefore also affect BMP-induced VEGF production of MSCsand negatively affect angiogenesis.

CONCLUSION

The inflammatory phase of fracture healing not only initiatesbut also directs downstream processes of bone repair, and dis-ruption of this phase by systemic inflammation or by local hy-perinflammation has been shown to impair fracture healing.The mechanism through which systemic inflammation impairsfracture healing, however, remains unknown.

As delineated in Fig. 1, we hypothesize that an increased influxof primed neutrophils into the fracture hematoma during sys-temic inflammation alters the composition of the fracture hema-toma and disturbs downstream processes of fracture healing. Thishypothesis is based on the facts that neutrophils form the major-ity of leukocytes, not only in the peripheral circulation but alsowithin the early fracture hematoma, and neutrophil numbers in-crease dramatically during systemic inflammation. Moreover, neu-trophils become primed during systemic inflammation, andprimed neutrophils are prone to home toward local sites of in-flammation. We believe that an increased influx of neutrophilimpairs fracture healing based on the findings that reduction ofsystemic neutrophils improves fracture healing, stimulates osteo-genesis, and inhibits chondrogenesis.

Although the potency of the fracture hematoma has gainedincreasing attention in the literature, the vast complexity ofprocesses that occur during the inflammatory phase of frac-ture healing still remains poorly understood. Additional re-search that addresses these processes will not only providetools to augment artificial cartilage and bone tissue generationbut may also contribute to the development of preventive ther-apies against risk factors of impaired fracture healing.

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KEY WORDS:leukocytosis � neutrophil � priming � bone � repair

Bastian et al. The effect of systemic inflammation on fracture healing

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