Differential gene expression analysis in fracture callus of patients with regular and failed bone healing

Injury, Int. J. Care Injured 43 (2012) 347–356

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Differential gene expression analysis in fracture callus of patients with regular and failed bone healing G. Zimmermann a,d,*, K.H.K. Schmeckenbecher a,d, S. Boeuf c, S. Weiss c, R. Bock c, A. Moghaddam b, W. Richter c a b c

Department of Traumatology and Orthopedic Surgery, Theresienhospital of the University of Heidelberg, Germany Department of Traumatology of the University of Heidelberg, Berufsgenossenschaftliche Unfallklink, Ludwig-Guttmann-Str. 13, 67071 Ludwigshafen, Germany Stiftung Orthopedic Clinic of the University of Heidelberg, Schlierbacher Landstr. 200a, 69118 Heidelberg, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Accepted 23 October 2011

Objective: Although several systemic and local factors are known to impair fracture healing, there is still no explanation, why some patients with sufficient fracture stability, showing none of the existing risk factors, still fail to heal normally. An investigation of local gene expression patterns in the fracture gap of patients with non-unions could decisively contribute to a better understanding of the pathophysiology of impaired fracture healing. For the first time, this study compares the expression of a large variety of osteogenic and chondrogenic genes in patients with regular and failed fracture healing. Methods: Between March 2006 and May 2007, a total of 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. Tissue samples of patients with normal and failed fracture healing were collected intraoperatively. Patients were divided into groups depending on the fracture date, and only patients with fractures two to four weeks old and patients with non-unions more than 9 months old were included in the final analysis. For the gene expression analysis, a customised cDNA array – containing 226 genes involved in osteo- and chondrogenesis – was used. Results: In the cDNA array analysis, the expression of eight genes was significantly elevated two-fold or more in the group with failed fracture healing relative to the normal controls. Conversely, no genes were found to be expressed at a higher level in the control group. The identified genes are supposed to be involved in extracellular matrix assembly, cytoskeletal structure, and differentiative and proliferative processes. Conclusions: The differences in gene expression pattern indicate a change in the composition and structure of the extracellular matrix, and a possible turn in the healing programme towards fibrous scar tissue formation, leading to non-union. ß 2011 Elsevier Ltd. All rights reserved.

Keywords: Non-unions Fracture healing Gene expression analysis Growth factors

Introduction Fracture healing is a physiological recovery process not comparable to any other tissue regeneration, leading to bone

Abbreviations: RT-PCR, reverse transcription polymerase chain reaction; bp, base pairs; ECM, extracellular matrix; SLRP, small leucine-rich repeat proteoglycan; EGF, epidermal growth factor; TGFb, transforming growth factor b; TNFa, tumor necrosis factor a; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of matrix metalloproteinases. * Corresponding author at: Theresienkrankenhaus Mannheim, Department of Traumatology, Bassermannstr. 1, 68165 Mannheim, Germany. Tel.: +49 621 4244432; fax: +49 621 4244654. E-mail addresses: [email protected], [email protected] (G. Zimmermann). d Equal contributors. 0020–1383/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.injury.2011.10.031

restoration of original quality and function.1–3 Further more, this healing process reflects embryonic development and bone growth.4–7 It is a highly specialised process combining many different biomechanical and biological factors. Every step to perfectly regenerated bone is controlled and regulated by various cytokines, growth factors, angiogenetic factors and proteolytic enzymes. If this sensitive balance is altered at any point, fracture healing will not occur properly. Many groups have investigated the process of bone regeneration. Today we know about several systemic and local factors that impair fracture healing. The healing process depends primarily on mechanical stability, which is determined by location and type of the fracture, width of the fracture gap, chosen osteosynthesis material, adaptation of the fragment ends and loading.8–11 However, certain biological conditions at the fracture site also

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contribute to the healing success, including soft tissue state, blood and oxygen supply, neuronal injury and bone infection. In addition, there are a number of systemic factors known to influence bone regeneration, such as anaemia, diabetes and hormone disorders.12– 14 Smoking and pharmacological treatment with steroids, nonsteroidal anti-inflammatory drugs, thromboprophylactic agents, cytostatics and antibiotics like fluoroquinolones and tetracyclines have also been discussed as possible causes for impaired fracture healing.13,15–18 However, there are still no sufficient explanations why some patients with sufficiently stabilised fractures, showing none of the above mentioned risk factors, still develop a non-union. Great efforts have been made to answer this question, using mostly in vitro studies and animal models or measuring systemic concentrations of various growth factors, angiogenetic factors and proteolytic enzymes in humans. An investigation of the local gene expression pattern in the fracture gap of patients with non-unions could decisively contribute to a better understanding of the pathophysiology of impaired fracture healing. Such a study has been carried out by Lawton and his group: they investigated tissue samples of the fracture gap in patients with normal and delayed fracture healing and compared gene expression of various mineralisation inhibitors in osteoblasts via in situ hybridisation and autoradiography.19 They found, that osteoblasts in slowly healing fractures express the gene encoding matrix gla protein, in contrast to those in regular bone healing. Motivated by these interesting results, we decided to analyse the expression of a wider range of genes in the fracture gap. Today microarray technology provides us with the possibility to analyse gene expression of a large number of genes simultaneously. This technology was successfully used to compare local gene expression pattern of normal fracture healing with non-unions in rats by Niikura et al.20 Gene expression levels of BMP (bone Morphogenic protein) -2, -3, -3B, -4, -6, -7, GDF (growth and differentiation factor) -5 and -7, as well as of BMP antagonists noggin, DRM, sclerostin, and BAMBI were significantly lower in non-unions compared to normally healing fractures at several time points. To our knowledge, no data on microarray analysis of pseudarthrosis in humans has been published so far. Our aim was to compare regular and failed fracture healing on the gene expression level and to elaborate possible differences. Materials and methods Patients and tissue samples Between March 2006 and May 2007, 130 patients who were surgically treated at the Berufsgenossenschaftliche Unfallklink Ludwigshafen were screened for the study. As inclusion criteria, we demanded patients to be over 18 years of age, and to give their formal consent, as well as radiological proof of long bone fracture. Because bone healing can be affected by various drugs and systemic disorders, patients suffering from renal insufficiency, liver disease, malignant tumours, collagenosis, inflammatory bowel disease, or haematological diseases were excluded from the study. Patients undergoing long-term treatment with steroidal or non-steroidal antiphlogistic drugs or other immunosuppressive agents, thromboprophylactic agents, fluoroquinolones and tetracyclines, and hormone substitution were also excluded. Smoking was not allowed for at least two months before surgical intervention. Patients were divided into different groups depending on the fracture date. As we wanted to compare gene expression in fresh callus tissue to pseudarthrosis tissue, only patients with fractures two to four weeks old and patients with real manifest non-unions more than 9 months old were included in the final analysis.

Table 1 Demographics of patients with normal fracture healing or non-unions, who were included in cDNA-array analysis. Non-unions

Unions Sex

Age

Local.

Sex

Age

Local.

f f m m m m m

80 77 32 66 24 18 32

Humerus Humerus Humerus Femur Femur Radius Radius

m m m m m m m f

60 54 44 65 42 42 44 39

Humerus Humerus Humerus Femur Femur Femur Tibia Tibia

f, female; m, male; local, fracture localization.

Demographics of the patients are presented in Table 1. All enrolled patients were followed up for a period of at least one year. The process of normal or failed fracture healing was confirmed by conventional X-ray, plain film tomography or computed tomography. Callus or pseudarthrotic tissue was collected out of the fracture gap during regular surgical treatment and stored at 80 8C. Samples measured approximately 10 mm  10 mm  10 mm. Biopsy collection did not extend operation time or increase risks for the patients. RNA extraction and cDNA array analysis Frozen tissue samples were pulverised mechanically and dissolved in lysis buffer for mRNA isolation using oligo-d(T)coupled beads (Dynabeads1; Dynal Biotech, Hamburg, Germany). For gene expression analysis, cDNA arrays were used, created in the research department of the Stiftung Orthopa¨dische Universita¨tsklinik Heidelberg, representing 226 genes involved in osteoand chondrogenesis and 4 housekeeping genes for signal normalisation.21 A list of all genes is represented in Appendix A (Table 3). mRNA isolated from tissue samples was reverse-transcribed to 33 P-labelled cDNA probes according to the manufacturer’s protocol (SuperScriptTMII; Invitrogen GmbH, Karlsruhe, Germany). The labelled cDNAs were denatured and hybridised to cDNA arrays in 7% SDS, 500 mM sodium phosphate buffer, pH 7.1 and 200 mg/ ml salmon sperm DNA overnight at 68 8C. Arrays were washed 2 15 min and 2  30 min in 1% SDS and 40 mM sodium phosphate buffer, pH 7.1 at 68 8C. Arrays were then exposed to a phosphor imaging plate for up to 72 h. Images were captured on a Bio-Imaging Analyser BAS-1800 II using BAS Reader 2.26 beta software (Fuji/Raytest, Straubenhardt, Germany). Spots were quantified with the Arrayvision software (Imaging Research Inc., St. Catharines, Ontario, Canada). Background-corrected intensity values were normalised and transformed to generalised ratios.22 Significance Analysis of Microarrays software (SAM) was used for comparison between gene expression patterns of normal and failed fracture healing.23 Background-corrected signal intensities normalised to the mean signal of each array were used for the calculation of fold changes. Results Of all patients included in the study, only those with fractures two to four weeks old and non-unions more than nine months old were taken for final analysis (Table 1). cDNA array analysis A total of seven callus tissue samples were compared to eight pseudarthrotic samples (Table 1). Samples were hybridised to a

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Table 2 List of genes with higher expression in samples from patients with non-unions. Mean expression level and ratio of gene expression values in pseudarthrosis patients in relation to controls are shown. Gene symbol

Gene name

Mean value non-unions

Mean value controls

Ratio

CDO1 PDE4DIP COMP FMOD CLU FN1 ACTA2 TSC22D1

Cysteine dioxygenase, type 1 Phosphodiesterase 4D interacting protein (myomegalin) Cartilage oligomeric matrix protein Fibromodulin Clusterin Fibronectin 1 Actin, alpha 2, smooth muscle, aorta TGF-b-stimulated protein TSC22 (TSC22 domain family, member 1)

3.79 8.46 21.84 18.29 33.16 47.83 14.61 8.74

0.25 1.64 4.41 3.86 8.06 16.54 5.91 4.00

15.37 5.15 4.95 4.73 4.12 2.89 2.47 2.18

customised 230 gene array and the pseudarthrosis and control groups were compared using the SAM software. With a false discovery rate set below 5%, we found eight genes in the group with failed fracture healing significantly more than two times higher expressed than in controls and no genes higher expressed in the controls (Table 2 and Fig. 1). Discussion Differences in gene expression between normal and failed fracture healing In this study, we were able to show a significantly different gene expression pattern in tissue samples from patients with regular fracture healing compared to samples from patients with nonunions. Eight genes could be identified with significant higher expression in pseudarthrosis than in the control group. Depending on their function, these genes can be divided into different groups. Extracellular matrix assembly and stability The correct composition and structure of the extracellular matrix (ECM) is essential for a tissue’s normal function and integrity. Of the genes, overexpressed in tissue samples from patients with pseudarthrosis, four appear to function in the assembly and stabilisation of the ECM: CDO1, COMP, FMOD and FN1. CDO1 encodes cysteine dioxygenase type 1, a cytosolic enzyme, found primarily in liver and brain, and to a lesser extent in kidney, heart and thyroid.24 This enzyme catalyses the oxygen-dependent conversion of Lcysteine into L-cysteinesulphinic acid, which is the first step in Lcysteine catabolism.25–27 CDO expression is stimulated through treatment with cysteine and inhibited through the cytokines TNFa and TGF-b.24 Cysteine is used for protein synthesis as well as for the synthesis of several non-protein compounds, including taurine, reduced inorganic sulphur, sulphate and glutathione. Together, these compounds are essential for a large number of critical functions in the body. In fact, the oxidative degradation of cysteine to inorganic sulphate is believed to be the major source of sulphate in vivo.24 Various conditions with autoimmune or inflammatory component, such as rheumatoid arthritis, systemic lupus erythematosus and primary biliary cirrhosis have been shown to be linked to high plasma levels of cysteine and low plasma concentrations of inorganic sulphate.24 As commonly known, sulphate is used for the side-chains of proteoglycans in the extracellular matrix. As the proteoglycans in the ECM of cartilage tissue mostly consist of chondroitin sulphate and keratan sulphate, CDO1 is likely to play an important role in the generation of the sulphate supply needed for ECM synthesis, and in so doing, it could also contribute to callus bone healing. Cartilage oligomeric matrix protein (COMP) is a non-collagenous protein, a pentameric member of the thrombospondin family, also called thrombospondin 5.28,29 It is expressed in a variety of

different cell types including chondrocytes, tenocytes, ligament cells, and osteoblasts.30 As this protein is easily detectable in serum, it has been investigated as a biomarker for cartilage processes in osteoarthritis.31 It exhibits a wide binding repertoire and has been shown to be involved in the regulation of chondrogenesis in vitro.28 Vitamin D3 and retinoic acid have been shown to bind within the central hydrophobic pore of its amino-terminal domain in vitro, suggesting a role for COMP as a storage and delivery protein for these molecules, which are important in bone development and metabolism.32 COMP-deficient-mice show no anatomic, histologic, or ultrastructural abnormalities.33 On the other hand, mutations in the COMP gene contribute to pseudoachondroplasia and multiple epiphysial dysplasias.34–37 COMP has been shown to bind to matrilins and types I, II, and IX collagen. It is therefore suggested to have a role in regulating fibril assembly, as well as a structural role in maintaining the mature collagen network, and it might also be involved in chondrocyte regulation.28 FMOD encodes fibromodulin, one of the small leucine-rich repeat proteoglycans (SLRPs).38 It is expressed in a variety of different tissues, with highest amounts in articular cartilage, tendon, and ligament.39 Their core proteins allow the SLRPs to interact with the fibrillar collagen that forms the framework of the tissue.38 They help regulate fibril diameter during its formation and possibly fibril– fibril interactions in the extracellular matrix. They also appear to limit access of collagenases to collagen molecules, and this way may protect the fibril from proteolytic damage. SLRPs have been reported to interact with many other macromolecules as well, including types VI, XII and XIV collagen, fibronectin and elastin, and growth factors such as EGF, TGF-b and TNF-a.38–40 By regulating growth factor access to the cells, the SLRPs may play a role in the modulation of chondrocyte metabolism. Fibromodulin knock-out mice show no change in appearance, but they display abnormal collagen fibril organisation in their tendons.41 FN1 encodes fibronectin 1. Fibronectins are a group of closely related glycoproteins encoded by a single gene, and the different proteins are produced following the alternative splicing of a single transcript.42 In the ECM, fibronectin is organised as an extensive network of elongated, branching fibrils. Fibronectins contain binding sites for different molecules, including sulphated glycosaminoglycans, DNA, collagen and gelatin. It is supposed, that the interaction of cells with fibronectin promotes actin organisation and cell contractility, cell growth, cell migration, cell adhesion, and ECM remodelling.43,44 Fibronectins also promote type I collagen deposition and enhances the tensile strength of collagen-based tissue constructs. They have been shown to affect MMP-9 function and angiogenesis.45,46 Fibronectin deficiency in humans has been identified in association with a form of Ehlers-Danlos syndrome.47 In chondrogenesis, fibronectin is thought to have a significant role in the differentiation of mesenchymal cells to chondral cells.48 It has been detected in greater amounts in osteoarthritic cartilage,

G. Zimmermann et al. / Injury, Int. J. Care Injured 43 (2012) 347–356

PDE4DIP

9 8 7 6 5 4 3 2 1 0

% hsk. Gene PA >9M

K 2-4W

18 16 14 12 10 8 6 4 2 0

45 40 35 30 25 20 15 10 5 0

PA >9M K 2-4W

FMOD

PA >9M K 2-4W

FN1

CLU

30

70

140

25

60

120

50

100

% hsk. Gene

% hsk. Gene

COMP

20 15 10

% hsk. Gene

% hsk. Gene

CDO1

% hsk. Gene

350

40 30 20

80 60 40

5

10

20

0

0

0

PA >9M K 2-4W

PA >9M K 2-4W

ACTA2

PA >9M K 2-4W

TSC22

35

20

25

% hsk. Gene

% hsk. Gene

30 20 15 10 5 0 PA >9M K 2-4W

15 10 5 0 PA >9M K 2-4W

Fig. 1. Expression levels of the identified genes in controls (2–4 weeks of healing) and non-unions (>9 months of healing). Expression values are presented in relation to the expression values of the housekeeping genes (% hsk gene). The horizontal bar represents the mean value.

possibly representing increased synthesis of proteins involved in matrix repair.49,50 In an experimental non-union rat model the formation of a fibrous bond, consisting mostly of type II collagen and fibronectin, was observed.48 This was interpreted as failure of the multipotentional mesenchymal stem cells to change the fracture healing process towards proliferation. Persisting instability and delay in mineralisation of the callus tissue seem to switch on a different healing cascade, with production of fibroblasts, type II collagen and fibronectin.

The overexpression of CDO1, COMP, FMOD and FN1 indicates that pseudarthrotic tissue shows a different assembly and structure of the extracellular matrix than normal healing callus. As atrophic non-unions are defined by a lack of callus tissue formation, this could be interpreted as regulatory mechanism to drive on the healing process and gain stability in the gap. This hypothesis however, would unavoidable lead to the question of, why only fibromodulin and not the other major cartilage proteoglycans show a higher expression in non-unions.

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Another interpretation could be that differences in ECM formation are the underlying cause of pseudarthrosis and are themselves due to a failure in the regulation of gene expression. This would be consistent with Hietaniemi’s suggestion of a switch in the bone healing programme towards fibrous scar tissue formation.26 Differentiation and proliferation During bone regeneration, numerous differentiative and proliferative processes take place in the fracture gap and are essential for healing success. It is therefore particularly intriguing, that amongst the identified genes overexpressed in pseudarthrotic tissue, there are two supposed to play an important role in this field. CLU has a nearly ubiquitous expression pattern in human tissues.51 Numerous cytokines, growth factors and stress or apoptosis inducing agents have been reported to regulate its expression. CLU encodes clusterin, a protein with two main isoforms, including a glycosylated secreted heterodimer (sCLU) and a truncated cytoplasmic/nuclear form (nCLU).52,53 Interestingly, these two isoforms seem to have completely opposing functions. The nuclear form of CLU protein is supposed to be proapoptotic, whereas the secreted form seems to be prosurvival. Clusterin contributes to numerous physiological processes, including development, differentiation and tissue remodelling.51,53,54 The CLU primer used in this experiment, detects the transcripts that encode both sCLU and nCLU. Therefore it is not clear whether only one of them is overexpressed in non-unions or if both are. TSC22 is a member of a family of leucine zipper transcription factors with repressor activity.55 It has been shown to homodimerise and exhibit transcriptional repressor activity when fused to a heterologous DNA-binding domain. It is also possible however, that TSC22 may modify gene expression through protein–protein interactions. It is expressed at varying levels in all foetal and adult tissues examined, except peripheral blood leukocytes.56 It is also an apoptosis-related gene that is upregulated in human epidermal keratinocytes after X-ray irradiation, and could function as a mediator of osteoarthritis.57,58 Since TGF-b stimulates TSC22 expression, one could assume that according to our findings, the systemic supply of this growth factor family should be increased in pseudarthrosis, leading to elevated local TSC22 levels in the fracture gap. However, compared to normally healing fractures, serum levels of TGF-b1 are significantly lower in patients with non-unions.3 Cytoskeleton The cytoskeleton is responsible for a cell’s structure and mechanical stability as well as for cell movement and intracellular transportation. The cytoskeletal gene ACTA2, which encodes the human aortic smooth muscle actin, is more than two fold higher expressed in tissue samples of patients with non-unions. Actin is the major component of microfilaments, and plays an important role in maintaining cell shape and movement. Smooth muscle a-actin is expressed in vascular smooth muscle cells and fibroblasts.59 Its expression is regulated by hormones and cell proliferation, and is altered by pathological conditions including oncogenic transformation. In non-unions, the overexpression of ACTA2 could be a marker for increased vascularisation in the area of the fracture gap. In this case, VEGF gene expression, which is a major regulating factor of angiogenesis, would be expected to be elevated as well.

351

On the other hand, as smooth muscle a-actin is also expressed in fibroblasts, this could mean, that pseudarthrotic tissue contains much higher amounts of this cell type than normally healing callus. This would correlate with the previously mentioned hypothesis of fibrous scar formation instead of bone restoration in the facture gap. PDE4DIP is another cytoskeletal gene, overexpressed in pseudarthrotic tissue samples. It encodes the phosphodiesterase 4D-interacting protein, also known as myomegalin, which was characterised because if its binding to the phosphodiesterase PDE4D, thereby targeting it to particulate structures.60,61 As the function of PDE4DIP remains mainly unclear, it is difficult to speculate about the significance of its overexpression in nonunions. It could play a role in intracellular targeting and thereby contribute to differentiative and proliferative processes taking place within the fracture gap. Conclusion Our aim was to compare normal callus tissue to pseudarthrosis tissue in humans on the gene expression level using cDNA arrays, scanning a large variety of genes, including those which have not been studied so far in the context of bone healing. As gene expression is a dynamic process changing according to the healing status of the tissue, we decided to choose two different time points for our study. Therefore we especially selected fractures 2–4 weeks old and real manifest non-unions more than 9 months old. Although the number of patients included is rather small, our data show significant differences in gene expression patterns of patients with regular and failed fracture healing. These differences indicate a change in the composition and structure of the extracellular matrix and a possible diversion in the healing programme towards fibrous scar tissue formation, leading to non-union. For some genes like COMP, FMOD and FN1 a close connection to cartilage tissue and, therefore, also to callous bone healing seems to be obvious. For others however, like TSC22, ACTA2 and PDE4DIP no studies have been carried out so far analyzing their role in bone tissue or fracture healing. Although we cannot proclaim a direct clinical relevance of our study, our aim was to contribute to a deeper understanding of the process of fracture healing and especially of the molecular mechanisms leading to non-unions. Therefore, to fully understand the role of the identified genes in this context, further investigation of their exact function and their relation to chondro- and osteogenesis would be necessary. To prove the theory of fibrous tissue formation in non-unions, gene expression profiling of this tissue type should be performed, with special emphasis on the occurrence of the genes discussed in this study. Also immunohistological staining for cartilage and connective tissue markers in non-unions could provide further information about the underlying mechanisms of pseudarthrosis. Conflict of interest statement All authors disclose any financial and personal relationships with other people, or organisations, that could inappropriately influence (bias) their work, all within 3 years of the beginning the work submitted. Acknowledgements This study was presented at the annual meeting for Orthopaedics and Trauma Surgery in Berlin, Germany, 2007 and at the bone biology congress in Berlin, Germany, 2008. The study was funded by a grant from the Orthopaedic University Hospital Heidelberg.

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Appendix A See Table 3. Table 3 List of all genes analysed. Clone ID is the gene identification on the cDNA array. Abbreviations correspond to the HGNC-database (Human Gene Nomenclature Database; www.gene.ucl.ac.uk/nomenclature/aboutHGNC.html). Clone ID

Genes

Abbreviation

ABCG ADAMTS14 ADAMTS16 ADAMTS5 ADAMTS9 AGC ALP ApM1 ARAB16 ARAB21 ARAB5 BGN BMPR1A BPAG1 BSP II C01t C02t C03t C04t C05t C06t C07t C08t C09t C10t C11t C12t C13t C14t C15t C16t C17t C18t C19t C20t C21t C22t C23t C24t C25t C26t C27l C28l C30t C31t C32t C33t C34t C35t C36t C37t C40l C41l C42l C44t C46t C47t C48t C49t C50t C51t C52t C53t C54t C56t C57t C58t C59t

ATP-binding cassette, sub-family G (WHITE), member 2 A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 14 A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 16 A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 5 A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 9 Aggrecan 1 Alkaline phosphatase, liver/bone/kidney Adipocyte, C1Q and collagen domain containing (adiponectin) Arabidopsis gene Arabidopsis gene Arabidopsis gene Biglycan Bone morphogenetic protein receptor, type IA Bullous pemphigoid antigen 1 Integrin-binding sialoprotein (bone sialoprotein, bone sialoprotein II) Hypothetical protein FLJ11151 ADP-ribosylation-like factor 6 interacting protein 2 AE (adipocyte enhancer) binding protein 1 early growth response 1 Rho GTPase activating protein 5 Ankyrin repeat and MYND domain containing 2 (hyp. protein DKFZp5640043) Chromosome 5 open reading frame 4 H19 gene Decorin Hypothetical protein FLJ 10707 Connective tissue growth factor Cysteine-rich protein 1 M96 protein, likely ortholog of mouse metal response element binding transcription factor 2 Hypothetical protein FLJ38101 Angiopoietin-like 2 Doublecortin domain containing 2 (KIAA1154 protein) Calsyntenin 1 Cysteine-rich protein 2 Hypothetical protein FLJ11526 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) B-factor, properdin mRNA, similar to rat phosphodiesterase 4D interacting protein (myomegalin) Cysteine dioxygenase, type I Similar to bK246H3.1 (immunoglobulin lambda-like polypeptide 1, pre-B-cell specific), FLJ32313 cDNA Clusterin Fibroblast growth factor receptor 3 CD74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) Shadow of prion protein Transforming growth factor beta-stimulated protein TSC-22 Surfeit 4 Proprotein convertase subtilisin/kexin type 1 inhibitor Integrin, alpha 10 Hypothetical protein LOC285733 RAB24, member RAS oncogene family Kell blood group GLE1 RNA export mediator-like (yeast) Aggrecan 1 (chondroitin sulphate proteoglycan 1) Protease, serine, 11 (IGF binding) Scrapie responsive protein 1 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 1 v-myb myeloblastosis viral oncogene homolog H factor 1 3-Hydroxyisobutyrate dehydrogenase MAX gene associated DKFZP564I1171 protein Glycophorin A Phospholipase A2, group IIA Tenascin XB FXYD domain containing ion transport regulator 6 Serine (or cysteine) proteinase inhibitor, clade A (alpha-1 antiproteinase, antitrypsin), member 3 Mitogen-activated protein kinase kinase kinase 7 interacting protein 1 SULF2 (similar to glucosamine-6-sulphatases) STE20-like kinase (yeast)

ABCG2 ADAMTS14 ADAMTS16 ADAMTS5 ADAMTS9 AGC1 ALPL ACDC ARAB5 ARAB16 ARAB21 BGN BMPR1A BPAG1 IBSP FLJ11151 ARL6IP2 AEBP1 EGR1 ARHGAP5 ANKMY2 C5orf4 H19 DCN FLJ10707 CTGF CRIP1 M96 FLJ38101 ANGPTL2 DCDC2 CLSTN1 CRIP2 FLJ11526 CD74, RASA4 BF PDE4DIP CDO1 LOC91316 CLU FGFR3 CD74 Sprn TSC22 SURF4 PCSK1N ITGA10 LOC285733 RAB24 KEL GLE1L AGC1 PRSS11 SCRG1 SERPINA1 MYB HF1 HIBADH MGA DKFZP 564I1171 GYPA PLA2G2A TNXB FXYD6 SERPINA3 MAP3K7IP1 SULF2 SLK

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Table 3 (Continued ) Clone ID

Genes

Abbreviation

C60t C61t C62t C63t C64t C65t C66o C67o C68o C69o C70o C71o C72o C73o C74t C75t C76l C77t C78t C80c C81c C82c C83r C84t C85o CAV1-1 Cbfa1 CD44 CD9 CDMP1 CDRAP CEP-68 CHAD CHM1 CILP COL10 COL11 COL12 COL1A1 COL2A1 COL3A1 COL9A1-1 COMP CRTL DCN ENG FMOD GAPDH ICAM1 IGFBP2 IGFBP5 IGFBP6 JNK-1 L1CAM LPL LUM M02t M03t M04t M05t M06s M07d M08o M09o M10o M11o M12o M13o M14d M15o M16o M17o M18o M19o M20o M21o M22d

Unknown chr. 16 Hypothetical protein LOC112868 Nedd-4-like ubiquitin–protein ligase (WWP2) S100 calcium binding protein, beta TGFB inducible early growth response 2 Matrix metalloproteinase 15 (membrane-inserted) CD59 antigen p18-20 (MACIF, MACIP) Ninjurin 1 Kangai 1 (suppression of tumorigenicity 6, prostate; CD82 antigen) Plasminogen activator, tissue Melanoma cell adhesion molecule Sialophorin (gpL115, leukosialin, CD43) Tumour necrosis factor receptor superfamily, member 7 Low density lipoprotein-related protein 1 (alpha-2-macroglobulin receptor) Frizzled-related protein Integrin, alpha L Contactin 1 Catenin (cadherin-associated protein), alpha 1, 102 kDa Ephrin receptor EphA1 Notch homolog 1, translocation-associated (Drosophila) Glycogen synthase kinase 3 alpha Lunatic fringe homolog (Drosophila) Ephrin-B3 Neural precursor cell expressed, developmentally down-regulated 4 Major histocompatibility complex, class I, C Caveolin 1 Runt-related transcription factor 2, core-binding factor-a1 CD44 antigen (hyaluronan receptor) CD9 antigen Cartilage-derived morphogenetic protein-1 (growth differentiation factor 5) Melanoma inhibitory activity (CD-RAP) Cartilage acidic protein 1 (CEP-68) Chondroadherin Leucocyte cell derived chemotaxin 1 (Chondromodulin I precursor) Cartilage intermediate layer protein Collagen, type X, alpha 1 Collagen, type XI, alpha 1 Collagen, type XII, alpha 1 Collagen, type I, alpha 1 Collagen, type II, alpha 1 Collagen, type III, alpha 1 Collagen, type IX, alpha 2 Cartilage oligomeric matrix protein Hyaluronan and proteoglycan link protein 1 (cartilage linking protein 1) Decorin Endoglin, CD105 Fibromodulin Glyceraldehyde-3-phosphate dehydrogenase Intercellular adhesion molecule 1 (CD54) Insulin-like growth factor binding protein 2 Insulin-like growth factor binding protein 5 Insulin-like growth factor binding protein 6 Mitogen-activated protein kinase 8 (C-Jun kinase 1) L1 cell adhesion molecule Lipoprotein lipase Lumican Tissue inhibitor of Metalloproteinase 1 Tissue inhibitor of metalloproteinase 3 Matrix metalloproteinase 1 Matrix metalloproteinase 2 Chondroitin sulphate proteoglycan 4 (melanoma-associated) (NG2) Natriuretic peptide precursor C Fibronectin 1 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide) Annexin A8 Twist homolog 1 (acrocephalosyndactyly 3; Saethre-Chotzen syndrome) Matrix metalloproteinase 9 (gelatinase B) Matrilin-1 Noggin Vascular cell adhesion molecule 1 Osteoprotegerin (tumour necrosis factor receptor superfamily, member 11b) Integrin a3 Laminin b1 Transglutaminase 2 Annexin A5 Matrix metalloproteinase 28 Matrix Gla protein

Unknown LOC112868 WWP2 S100B TIEG2 MMP15 CD59 NINJ1 KAI1 PLAT MCAM SPN TNFRSF7 LRP1 FRZB ITGAL CNTN1 CTNNA1 EPHA1 NOTCH1 GSK3A LFNG EFNB3 NEDD4 HLA-C CAV1 RUNX2 CD44 CD9 GDF5 MIA CRTAC1 CHAD LECT1 CILP COL10A1 COL11A1 COL12A1 COL1A1 COL2A1 COL3A1 COL9A2 COMP HAPLN1 DCN ENG FMOD GAPD ICAM1 IGFBP2 IGFBP5 IGFBP6 MAPK8 L1CAM LPL LUM TIMP1 TIMP3 MMP1 MMP2 CSPG4 NPPC FN1 ITGA5 ANXA8 TWIST1 MMP9 MATN1 NOG VCAM1 TNFRSF11B ITGA3 LAMB1 TGM2 ANXA5 MMP28 MGP

G. Zimmermann et al. / Injury, Int. J. Care Injured 43 (2012) 347–356

354 Table 3 (Continued ) Clone ID

Genes

Abbreviation

M23o M24o M25o M26d M27t M28l M29t M30t M31t M33c M34c M35c M36c M37c M38c M39c M40r M41c M42c M43c M44r M45l M46r M47c M48c M49c M50c M51c M52c M53r M54c MMP10 MMP13-1 MMP16 MMP3 MMP8 MRC2 NLK OB OC ON OPN PiT1

A disintegrin and metalloproteinase domain 15 (metargidin) Hypoxia-inducible factor 1, alpha subunit Syndecan 3 CD36 antigen (collagen type I receptor, thrombospondin receptor) Fibrillin 1 Nerve growth factor receptor (TNFR superfamily, member 16), p75 Tenascin C Calcitonin receptor-like receptor Matrilin 3 Perlecan, heparan sulphate proteoglycan 2 Nitric oxide synthase 2A (inducible, hepatocytes) Actin, alpha 2, smooth muscle, aorta Tissue inhibitor of metalloproteinase 4 Cathepsin B Integrin b1 A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 12 Collagen, type XIV, alpha 1 (undulin) Matrix metalloproteinase 11 (stromelysin 3) AXL receptor tyrosine kinase Collagen, type VI, alpha 1 Cadherin 2, type 1, N-cadherin (neuronal) A disintegrin-like and metalloprotease with thrombospondin type 1 motif, 1 Integrin av Hyaluronan synthase 1 Neural cell adhesion molecule 1 Thrombospondin-1 Growth arrest-specific 6 Tissue inhibitor of metalloproteinase 2 b catenin (cadherin-associated protein), beta 1 Fibulin 1 Vascular endothelial growth factor Matrix metalloproteinase 10 (stromelysin 2) Matrix metalloproteinase 13 (collagenase 3) Matrix metalloproteinase 16 (membrane-inserted) Matrix metalloproteinase 3 (stromelysin 1, progelatinase) Matrix metalloproteinase 8 (neutrophil collagenase) Mannose receptor, C type 2 (endo 180) Glucose phosphate isomerase (neuroleukin) Leptin (obesity homolog, mouse) Bone gamma-carboxyglutamate (gla) protein (osteocalcin) Secreted protein, acidic, cysteine-rich (osteonectin) Secreted phosphoprotein 1 (osteopontin, bone sialoprotein I) POU domain, class 1, transcription factor 1 (Pit1), solute carrier family 20 (phosphate transporter), member 1; sodium dependent Pi transporter typ III Proline arginine-rich end leucine-rich repeat protein Superficial zone protein (proteoglycan 4) Parathyroid hormone receptor 1 Parathyroid hormone related protein (PTH-like hormone) Focal adhesion kinase pp125 (tyrosine kinase) Erythrocyte membrane protein band 4.1-like 3 Natriuretic peptide receptor A/guanylate cyclase A (atrionatriuretic peptide receptor A) Polo-like kinase 1 (Drosophila) Aurora kinase B Interleukin 13 receptor, alpha 2 Fatty acid binding protein 3, muscle and heart (mammary-derived growth inhibitor) Integral membrane protein 2A UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-like 1 Lamin A/C Ribosomal protein L13a Ribosomal protein S9 Thymosin, beta 10 Polymerase (RNA) II (DNA directed) polypeptide L Transgelin Calumenin Hypothetical protein FLJ21986 Hypothetical protein FLJ90652 Lectin, galactoside-binding, soluble 1 (galectin 1) Chromosome 1 open reading frame 29, mRNA expr. In osteoblasts Serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1 Apoptosis inhibitor 5 (FGF2-interacting factor, XAGL protein) S100 Calcium binding protein A11 (Calgizzarin) IDN3 protein D component of complement, adipsin Adrenomedullin Mitochondrial gene, 16 S rRNA KIAA0746 protein

ADAM15 HIF1A SDC3 CD36 FBN1 NGFR TNC CALCRL MATN3 HSPG2 NOS2A ACTA2 TIMP4 CTSB ITGB1 ADAMTS12 COL14A1 MMP11 AXL COL6A1 CDH2 ADAMTS1 ITGAV HAS1 NCAM1 THBS1 GAS6 TIMP2 CTNNB1 FBLN1 VEGF MMP10 MMP13 MMP16 MMP3 MMP8 MRC2 GPI LEP BGLAP SPARC SPP1 POU1F1

PRELP PRG4 PTHR1 PTHrP PTK2 RDA F1H RDA F2D RDA F2F RDA F4F RDA F5C RDA F5F RDA F7E RDA F8C RDA MA2 RPL13A RPS9 S01b S03t S04l S05t S06t S07t S08t S10t S11t S12l S13t S14t S15t S16t S18t S19t

PRELP PRG4 PTHR1 PTHLH PTK2 EPB41L3 NPR PLK1 AURKB IL13RA2 FABP3 ITM2A GALNTL1 LMNA RPL13A RPS9 TMSB10 POLR2L TAGLN CALU FLJ21986 FLJ90652 LGALS1 C1orf29 SERPINF1 API5 S100A11 IDN3 DF ADM MTRNR2 KIAA0746

G. Zimmermann et al. / Injury, Int. J. Care Injured 43 (2012) 347–356

355

Table 3 (Continued ) Clone ID

Genes

Abbreviation

S20t S21t S22t S23t S25t S26t S27t S28t SOX9 ß-Aktin STAG VER YKL39 YKL40 ZYX1-1

Hypothetical protein 628 (LOC56270) Tropomyosin 2 (beta) Laminin receptor 1 (ribosomal protein SA, 67 kDa) COP9 constitutive photomorphogenic homolog subunit 8 (Arabidopsis) Unknown chr. 12 Four and a half LIM domains 2 Syntaxin binding protein 2 Huntingtin interaction protein 1 SRY (sex determining region Y)-box 9 Actin, beta Stromal antigen 1 Chondroitin sulphate proteoglycan 2 (versican) Chitinase 3-like 2 (YKL-39) Chitinase 3-like 1 (cartilage glycoprotein-39; YKL-40) Zyxin 2

LOC56270 TPM2 LAMR1 COPS8 Unknown FHL2 STXBP2 HIP1 SOX9 ACTB STAG1 CSPG2 CHI3L2 CHI3L1 ZYX

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