Acquiring Tetanus After Hemorrhoid Banding and Other Gastrointestinal Procedures

Tetanus after hemorrhoidal banding is an extremely rare but serious complication of the procedure. We describe the second reported case of this complication and review the literature concerning tetanus after different gastrointestinal procedures. Although a rare complication, practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in at-risk patient populations. Such cases also reemphasize the importance of primary tetanus immunization and follow-up boosters for all vulnerable patients.
Introduction
Tetanus is currently a rare disease in the United States and other developed countries where active immunization programs provide a considerable degree of immunity to the infection in the general population. The incidence of tetanus has dropped dramatically in the United States and other developed countries due to a successful vaccination program instituted in the 1940s.1 During the period from 1998 to 2000, the Centers for Disease Control (CDC) reported the average annual incidence of tetanus to be 43 new cases in the United States per year. This is equivalent to 0.16 cases/million people in the US.2 The worldwide incidence of tetanus, however, is much higher and estimated to be around 500,000 to 1 million per year.3 In developed countries, tetanus is most commonly seen in immigrant patients who have never received a complete primary immunization series. Another at-risk group is the elderly people who have not received recent booster doses. Such cases in the older, previously vaccinated adult reflects waning immunity if a booster shot is not received every 10 years.
Tetanus is caused by the toxin-producing anaerobic bacterium Closteridium tetani. C. tetani is a spore-forming bacteria that once inside the body of susceptible host, produces a potent toxin named tetanospasmin. Tetanospasmin binds to the central nervous system causing diffuse muscle spasms and autonomic instability that characterizes tetanus. Tetanus is usually seen in association with soil contamination of a cutaneous wound; however, on rare occasions it may occur after a surgical procedure such as gastrointestinal surgery. In this paper, we describe the second reported case of tetanus after banding ligation of internal hemorrhoids and will review the other reported cases of tetanus after gastrointestinal procedures.
Case Report
A 63-year-old female with no significant past medical history presented with chief complaint of 5 days of anal discomfort due to hemorrhoids. Physical exam showed a 1.5 cm soft and inflamed external hemorrhoid. She was initially treated with pramoxine suppositories and a week later was scheduled for elective hemorrhoid banding. At the time of surgery, she was noted to have a small noninflamed external hemorrhoid as well as a grade III internal hemorrhoid at the eight o’clock position; there was no evidence of infection. The patient underwent rubber band ligation of the internal hemorrhoid with no immediate complications. After the procedure, the patient was instructed to take daily Sitz baths and given a prescription for docusate (oral) and psyllium to prevent constipation. Five days after the surgery, the patient returned to the hospital with a 1-day history of sore throat, pain upon swallowing, headache, and inability to open her mouth. She was noted to have a hoarse voice and stated that she had difficulty sticking out her tongue. Initial vital signs showed blood pressure of 150/70 mmHg, pulse of 95, temperature of 37.1°C, and respiratory rate of 18 per minute. Neurological examination was remarkable for inability to completely open the mouth; the rest of the neurological examination showed normal findings including muscle tone, bulk, and strength. A neck x-ray was obtained and it was normal. Patient’s inability to open mouth prompted further evaluation and the diagnosis of tetanus was entertained. Further history revealed that patient had never been immunized against tetanus. She was subsequently admitted to the intensive care unit and initially treated with 1,000 units of intervenous (IV) human tetanus immune globulin, 2 g of IV ceftriaxone every 24 h, IV diazepam, and methylprednisolone. On the next day, the patient had severe pain on attempting to open her mouth and diffuse muscular spasms of jaw, neck, abdominal musculature consistent with trismus and generalized tetanus. A rectal exam at the time showed normal postoperative changes; there were no masses, bleeding, signs of infection, or hemmorhoids. She subsequently developed respiratory difficulties due to laryngeal spasms and required intubation and paralysis. In the next days, she developed a number of complications related to hospitalization and the underlying tetanus. The patient had autonomic lability with associated hypertension (systolic blood pressure up to 220 mmHg) and tachycardia (pulse of 110s); she developed an episode of chest paint and was found to have an acute anterior wall ST-elevation myocardial infarction confirmed with EKG abnormalities and elevated troponins. Additional complications included a small pneumothorax after placement of a Swann–Ganz catheter, left lung collapse due to mucous plugging, and nosocomial pneumonia due to Acinetobacter baumanii. Attempts to wean her during endotracheal intubation were unsuccessful and the patient required a tracheostomy. After about 2 months of hospitalization, she gradually improved and was weaned from the respirator. She had upper and lower muscle atrophy, global muscle weakness, and ankle contractures bilaterally. She had no obvious sensory, cognitive, or language deficits. She was able to roll herself in bed and feed herself; however, she was unable to stand, walk, or place herself on bedpan. She was subsequently transferred to an inpatient rehabilitation facility for intensive physical and occupational therapy. At the completion of a month-long inpatient rehabilitation program, she showed some improvement but still had some lower extremity weakness (grade 3–4 out of 5 of muscle strength bilaterally) and residual ankle contractures; she could walk with assistance using a forearm crutch. The patient was discharged from the hospital with a continuing outpatient rehabilitation program.
Review of Literature
Methods
We searched the English language articles from 1966 to January 2005 in the MeSH system of PubMed for relevant case reports and articles. MeSH keywords identified included tetanus, combined with digestive system surgical procedures, surgery, surgical procedures operative, and hemorrhoids. All the relevant articles were reviewed, and their reference list examined for other relevant articles. Other articles were obtained and reviewed from these reference lists. Postoperative tetanus cases after obstetric–gynecology procedures in which the appendix was removed were not included; one such paper is included in the reference list however.4
Results
We found 14 case reports of tetanus after gastrointestinal procedures. Findings and case summaries are outlined in the tables below (see Tables 1 and 2).
Table 1Reported Cases of Gastrointestinal Procedures Complicated by TetanusCasesReferencesOpen cholecystectomy5–7Cholecystectomy with exploration of bile duct8Resection for a gangrenous perforated small intestine9,10Rubber band ligation of hemorrhoids11Cryosurgery for internal hemorrhoids12Drainage of anorectal abscess13Sigmoidoscopic polypectomy14Gastrectomy, Billroth II, and transverse colectomy, (for large cell lymphoma)15Exploratory laparotomy (for carcinoma in omentum and liver)5Table 2Summary of Presentations for the Reported CasesAuthorYearAge (Year)Gender (Male/Female)Time to Initial Symptoms. (Days Postprocedure)Initial SymptomsLater SymptomsOnset of Later Symptoms (Days Postprocedure)Tetanus ImmunizationOutcomeOpen cholecystectomy: (four cases) 5–7Parker& Mandal 5198447Female10No mentionTrismus10uncertainAliveParker& Mandal 5198459Female17Spastic gaitNo mention22No mentionDeadO’Riordain, Buckley, & Kirwan 6199146Male11Abdominal spasm, pain, fever, mild trisumusTrismus12NoDeadCrokaert, Glupczynski, Fastrez, Alle, & Yourassowsky 7198444No mention6Neck and face pain, stiffnessOpisthotonus6No mentionNo mentionCholecystectomy with exploration of bile duct: (two cases) 8Lennard, Gunn, Sellers, & Stoddart 8198449Female12Sub-costal pain, abdominal distentionOpisthotonusNo mentionNoAliveLennard et al. 8198466Female16Abdominal painTrismus19NoAliveResection for a gangrenous perforated small intestine: (two cases) 9,10Furui et al. 9199875Male1Jerking, limb rigidityOpisthotonus1NoAliveClay & Bolton 10196461Male2JerkingOpisthotonus4No mentionAliveRubber band ligation of hemorrhoids: (two cases) 11Murphy 11197833Female7Dysphagia, neck painTrismus9NoAlivePresent case199763Female4DysphagiaTrismus6NoAliveCryosurgery for internal hemorrhoids: (one case) 12Singh, Chhina, & Kaul 12199242Male14Fever, DysphagiaTrismus19NoAliveDrainage of anorectal abscess: (one case) 13Myers et al. 13198462Male10Restlessness, limb rigidityTrismus12NoAliveSigmoidoscopic polypectomy: (one case) 14Segel & Shaff 14196955Female10Fatigue, weaknessTrismus19NoAliveGstrecotmy, Billroth II, and transverse colectomy, for “large cell lymphoma”: (one case) 15Fleshner, Hunter, & Rudick 15198848Male21Fever, abdomen, back pain, dysphagiaTrismus and opisthotonus26NoAliveExploratory laporatomy (for carcinoma in omentum and liver): (one case) 5Parker & Mandal 5198465Female12No mentionNo mention12NoDead
Discussion
This is the second case of tetanus after hemorrhoid banding that we were able to find in the literature. As noted in Table 1, tetanus can occur after a wide variety of gastrointestinal surgical procedures including major intraabdominal surgery (e.g., cholecystectomy for cholecystitis; exploratory laparotomy for intestinal perforation), as well as relatively “trivial” procedures such as hemorrhoid surgery and sigmoidoscopic polypectomy. Tetanus occurs after germination of the spores and subsequent production of toxin by the organism. Clostridium tetani will not grow in healthy tissue, therefore a number of factors need to be present for germination to occur including ischemia, devitalized tissue, coinfection, and injury from penetration or foreign body.1 In gastrointestinal surgical procedures, presence of ischemic or devitalized tissues permits proliferation of C. tetani and subsequent toxin production. In our case, the devitalized tissue from banded hemorrhoid was the likely entry point for the organism with subclinical C. tetani infection occurring at the site. C. tetani may be isolated from stool flora in asymptomatic individuals; in farming regions where individuals are in constant contact with soilor domestic animals (e.g., horses, cattle), up to 1/3 of individuals may have C. tetani in their stools.16 Given standard sterilization procedures, tetanus associated with infected surgical instruments is extremely uncommon in industrialized countries—our patient most likely developed the condition after contamination of the wound with organisms from the stool or external environment. In our experience with patients from Latin America, it has come to our attention that some of them use herbal products such as out-door plants known as aloe vera (“buena herba” in Spanish) for treatment of local wounds. Although we are not aware of any documented case report of this practice causing transmission of tetanus, we wonder if application of contaminated outdoor plants might play a role in some of the postoperative cases. Our patient did not provide history of such practice and the exact mode of contamination will not change the clinical presentation or management. Once suspicion arises for presence of tetanus, identification depends upon recognition of the characteristic clinical syndrome. Although isolation of C. tetani from a wound is supportive of the diagnosis, many patients have negative wound cultures and presence of the characteristic clinical syndrome is adequate evidence to support the diagnosis.1
Although rare, it is prudent for practitioners to be mindful of signs that suggest the possibility of postoperative tetanus. Patients with tetanus usually have painful spasms and contractions of their skeletal muscles; this can present as stiff neck, trismus, or opisthotonus. Lockjaw, also known as trismus, leads to inability to open the mouth and the characteristic sardonic smile (risus sardonicus)—it is a sign of spasm of muscles of mastication. Trismus, along with back pain and diffuse muscle spasms, is one of the most frequent findings in tetanus.17 Opisthotonus is a sign of generalized tetanus and is characterized by painful involuntary bending of spine and extremities. It leads to forward convexity of the body, with patient’s torso arching upward and body supported on head and heels. There can also be periods of apnea due to thoracic, glottal, pharyngeal muscle contractions often requiring intubation and respiratory support. Irritability, restlessness, sweating, labile vitals signs, or even a myocardial infarction can occur due to autonomic instability. Presence of these signs or symptoms could be due to underlying tetanus and should alert the practitioner of possibility of the condition. Despite the case reports in Table 1, it should be emphasized that tetanus is a highly unusual complication of gastrointestinal surgical procedures. Given the extremely rare incidence of this complication, it does not appear to be practical to require evidence of full tetanus immunization before a procedure. Nevertheless, our case reemphasizes the importance of routine tetanus immunization, especially in patient populations that were not immunized in childhood or failed to receive a complete series of vaccinations. As part of their general health maintenance, adults should continue to receive periodic booster shots every 10 years as recommended by public health authorities.18 Furthermore, despite its rarity, practitioners need to be aware of the clinical presentation of tetanus and consider the diagnosis in at-risk patients who present with characteristic symptoms such as trismus and muscle rigidity.
Conclusion
Postoperative cases of tetanus have been reported after a number of different gastrointestinal procedures. Tetanus can occur after relatively minor procedures such as hemorrhoidal banding. Practicing physicians need to be aware of the clinical presentation of this deadly disease when encountered in vulnerable patient populations.

tetanus
hemorrhoids
digestive system surgical procedures
surgical procedures operative
postoperative complications


Analysis of obstetric complications and uterine connective tissue in tenascin-X-deficient humans and mice

Tenascin-X (TNX) is a large, multi-domain, extracellular matrix glycoprotein. Complete deficiency of TNX in humans leads to a recessive form of Ehlers-Danlos syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Here, we present a study of genitourinary and obstetric complications in TNX-deficient women of reproductive age. We have found complications, such as uterus prolapses, that are in agreement with previous findings in other EDS types. In TNX knockout (KO) mice, we have observed mild pregnancy-related abnormalities. Morphological and immunohistological analysis of uterine tissues has not revealed obvious quantitative or spatial differences between TNX KO and wildtype mice with respect to collagen types I, III, V, and XII or elastic fibers. We conclude that TNX-deficient women are at risk of obstetric complications, but that TNX KO mice show only a mild phenotype. Furthermore, we show that TNX is involved in the stability of elastic fibers rather than in their initial deposition.
Introduction
Tenascin-X (TNX) is a large, multi-domain, extracellular matrix (ECM) glycoprotein composed of epidermal growth factor (EGF) like-repeats, fibronectin type III repeats, and a C-terminal fibrinogen domain (Bristow et al. 1993; Lethias et al. 1996; Elefteriou et al. 1997; Ikuta et al. 1998; Tucker et al. 2006). Complete deficiency of TNX in humans leads to a rare recessive form of Ehlers-Danlos Syndrome (EDS), and TNX haploinsufficiency is a cause of hypermobility type EDS. Patients of both EDS types exhibit mild to severe joint hypermobility. The skin of TNX-deficient patients is markedly lax with poor recoil properties and shows easy bruising. The collagen density appears reduced in the dermis of these patients, and the elastic fibers are abnormal (Burch et al. 1997; Schalkwijk et al. 2001; Zweers et al. 2003; Peeters et al. 2004; Lindor and Bristow 2005). Most of the literature concerning pregnancy in EDS uses the old classification of nine subgroups. In our study, we use the revised classification of six subtypes of EDS (Beighton et al. 1998). Tissue fragility is a hallmark of EDS and is present in all the different subtypes. EDS patients appear to have a higher risk of several complications during pregnancy, such as pelvic instability, premature rupture of membranes, and postpartum hemorrhage. Pregnancy in classical and hypermobility type EDS has a relative favorable maternal and neonatal outcome. Pregnancy in vascular type EDS patients is associated with severe complications including maternal mortality (Pepin et al. 2000; Carley and Schaffer 2000; Roop and Brost 1999; Wegrowski et al. 1999; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998). The effect of TNX deficiency on pregnancy has not been studied so far, and only one case report of pregnancy in a TNX-deficient woman has been reported in the literature (Lindor and Bristow 2005). Here, we present a study of all known pregnancies and genito-urinary (GU) abnormalities in TNX-deficient woman of reproductive age identified in our clinic (Schalkwijk et al. 2001) and in the literature (Lindor and Bristow 2005; Schalkwijk et al. 2001). Furthermore, we have extended our studies to a TNX knockout (KO) mouse model in which we have investigated uterine development during pregnancy. We have previously established that TNX is essential for the development of tissue strength in the skin and is able to bind to elastin, a major component of elastic fibers, and to collagens type I, III, V, and XII (Egging et al. 2006a, b; Veit et al. 2006). We have investigated the expression of these molecules at the protein level.
Materials and methods
TNX-deficient patients
We investigated pregnancies and GU abnormalities in all currently identified female tenascin-X deficient patients of reproductive age identified in our clinic and in the literature. The study protocol was approved by the local medical ethics committee, and written informed consent was obtained from the patients.
Experimental animals
TNX knockout (KO) mice were obtained as described previously (Mao et al. 2002). For all studies, we used TNX KO mice that were crossed back with six generations of C57BL/6N mice. Wildtype C57BL/6 (WT) mice were used as a control. All mice investigated for pregnancy abnormalities and morphological changes in uterus tissue were between 2 and 6 months of age, reflecting a normal age distribution of a breeding mice population. Skin samples were taken from 2-month-old and 9-month-old mice, and aortic tissue was obtained from 9-month-old mice. The experimental design was approved by the Animal Use Committee of Radboud University, Nijmegen.
Breeding
Breeding pairs were kept in a 5:1 female:male ratio. Females were inspected daily for vaginal plugs, and those with plugs were isolated and inspected each day for abnormalities and progression of pregnancy. Litter size was determined after birth. Neonatal survival is defined as the difference in litter size after partus and 2 weeks postpartum.
Processing of samples for (immuno)histochemistry
Mice were killed in a sealed compartment by exposure to a mixture of carbogen gas and increasing concentrations of CO2. Skin samples for frozen sections were embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe, The Netherlands) and snap frozen in liquid nitrogen. Skin samples for paraffin sections were fixed in 4% buffered formalin for 4 h.
Affinity-purified TNX antibody production
TNX FNIII repeats 27–32 were amplified by polymerase chain reaction (PCR; forward primer: 5’-GGAATTCGAGCTACCTCCCCAC-3’, reverse primer: 5’-CAGGTCGACTCAGGTGAAAGAGGTGGA-3’) by using a previously described 2.7-kb human TNX cDNA as a template (Morel et al. 1989). The PCR product was ligated into the pCR2.1TOPO vector (Invitrogen, Breda, The Netherlands) according to the manufacturer’s instructions for easy digestion with restriction enzymes. The pCR2.1 TOPO vector with insert was digested with EcoRI and SalI. The region coding for FNIII27-32 was inserted into the EcoRI/SalI site of the pET28(a)+ plasmid (Brunschwig Chemie, Amsterdam, The Netherlands). The sequence of the TNX domains was verified by dideoxy sequencing with a 3730 DNA analyzer (Applied Biosystems, Nieuwekerk a/d IJssel, The Netherlands). TNX FNIII27-32 protein was expressed and purified according to the manufacturer’s instructions (Brunschwig Chemie). Purified TNX FNIII27-32 protein was used for immunization of a rabbit. Aliquots of 500 μg TNX FNIII27-32 in 500 μl phosphate-buffered saline were mixed with an equal volume of Freund’s complete adjuvant for the first injection and Freund’s incomplete adjuvant for boosters. Three injections were administered subcutaneously at 3-week intervals. Polyclonal antibodies against TNX FNIII27-32 were purified by affinity chromatography on a column with the antigen coupled to CNBr-activated sepharose 4B (GE Healthcare Life Sciences, Diegem, Belgium).
Histopathology, histochemistry, and immunohistochemistry
Staining of elastic fibers was performed by modified Hart’s stain on paraffin sections (7 μm; Starcher et al. 2005). Paraffin sections were also stained with hematoxylin and eosin (H&E) stain to assess uterus morphology and pathology, in collaboration with a professional pathologist. Frozen sections (7 μm) were stained with antibodies against collagen types I, III, and V (SouthernBiotech, USA) and elastin (Elastin Products, USA) as previously described by Egging et al. (2006b). Collagen XII was stained as previously described by Veit et al. (2006). TNX was stained with affinity-purified antibodies directed against FNIII27-32 of TNX. Detection was performed with antibodies labeled with fluorescein isothiocyanate (FITC, green; DakoCytomation, Denmark) and Alexa Fluor 594 (AF594, red; Molecular Probes, The Netherlands). Cell nuclei were made visible with 4,6-diamidino-2-phenylindole (DAPI, blue; Molecular Probes) counterstaining. Assesment of immunostaining was performed as previously described (Egging et al. 2007, 2006b). Images were captured and processed with Axiovision software (Carl Zeiss, The Netherlands).
Electron microscopy
For electron microscopy, WT and TNX KO samples from uterine wall at 3 weeks post-partum, skin from 2-month-old and 9-month-old mice, and aortas from 9-month-old mice were fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate overnight and washed in 0.1 M sodium cacodylate buffer. The tissues were then sequentially treated with osmium tetroxide, tannic acid, and uranyl acetate and then dehydrated and embedded in Epon as previously described (Davis 1993). Thin sections (60 nm) were counterstained with 7% uranyl acetate in absolute methanol and lead citrate and examined in a Tecnai 12 transmission electron microscope, at 120 kV.
Statistics
For statistical analyses, Fisher’s exact or Chi-square tests were used where appropriate for proportional data. Student’s t-test was used to compare mean litter size after partus and neonatal survival (difference in litter size after partus and 2 weeks postpartum). A P-value of <0.05 was considered statistically significant.
Results
Pregnancy in TNX-deficient patients
We investigated pregnancy and GU abnormalities in TNX-deficient woman of reproductive age. General reproduction characteristics and GU abnormalities of female TNX-deficient patients are presented in Table 1. Maternal and neonatal outcome was generally normal; one out of 13 pregnancies resulted in an intrauterine death of the fetus. Tissue laxity of TNX-deficient patients was demonstrated by the occurrence of vaginal, uterine, and rectal prolapses, even at a relative young age. One case of postpartum hemorrhage was observed in 12 childbirths. None of our patients had urinary incontinence symptoms. TNX-deficient EDS is an autosomal recessive disorder; therefore, all offspring of a TNX-deficient patient and an unaffected individual are obligatory heterozygotes. We have previously found that approximately 60% of adult females that are haploinsufficient for TNX suffer from hypermobility type EDS or benign joint hypermobility syndrome (Zweers et al. 2003). We found no obvious abnormalities (e.g. floppy infant syndrome, premature birth) in the neonates (obligatory heterozygotes) of our TNX-deficient population; however, some TNX-deficient patients themselves were born prematurely (Table 1; Lindor and Bristow 2005). Four out of six of the TNX-deficient women had postpartum complications (one of the patients had no pregnancies). This was in agreement with the findings in the literature of an increase in maternal GU complications in EDS-deficient women compared with the control population (Ainsworth and Aulicino 1993; Carley and Schaffer 2000; Kuczkowski 2005; Lind and Wallenburg 2002; Ramos-e-Silva et al. 2006).
Table 1Pregnancy in TNX-deficient woman (VUE vaginal uterine extirpation, IUD intra-uterine death, CAH congenital adrenal hyperplasia). Age represents the age of the patient at the anamnesisPatient IDAge (years)Gravida number/Para numberGenito-urinary complicationsSpecification150G1P1NoneNone2 (sister of patient 1)60G2P2 VUE after uterine prolapse (at age 49)None Vaginal prolapse 2 weeks after VUENone346G0P0NoneWas born prematurely (week 35) with shoulder luxation, rectal prolapse at age 1 4 (sister of patient 3)51G3P3 One incident of hemorrhage postpartum (>1000 ml blood loss)None538G0P0NoneCAH 651G3P2 IUD (24 weeks) with relatively large blood lossDeceased at age 51, severe co-morbidity including cardiomyopathy, arrhythmia, arteriosclerosis, and amputation of left leg (Schalkwijk et al. 2001). Undefined prolapse (at age 36)757*G4P4 Precipitious second stage at term for all birthsLindor and Bristow 2005 Uterine prolapse (at age 20), also recurrent rectal prolapses
Pregnancy in TNX KO mice
We investigated GU and pregnancy-related abnormalities in TNX KO and WT mice ranging between 2–6 months of age. Litter size (TNX KO: 7±2 pups, WT: 7±3 pups) and neonatal survival (TNX KO: 74%±36%, WT: 54%±40%) did not differ significantly (Student’s t-test). GU abnormalities of the TNX KO and WT mice are presented in Table 2. Overall, we noted relatively few pregnancy-related abnormalities. Obstruction of the uterine or vaginal canals during partus resulting in a failure to deliver pups was noted more often in WT mice compared with TNX KO mice, but the difference was not significant (Chi-square test). The length of term did not differ significantly for the TNX KO mice (mean: 19.3 days) compared with WT mice (mean: 19.6 days, Fisher’s exact test). A striking difference, however, between TNX KO and WT was found for the location of the vaginal plug that was present after mating. Generally, the vaginal plug in TNX KO mice was located much deeper in the vaginal canal than in WT mice. This made identification of vaginal plugs in TNX KO mice markedly more difficult, resulting in a significant increase in pregnancies with unknown length of pregnancy (Fig. 1a, Chi-square test). No uterine or rectal prolapses were observed in this particular study (67 pregnancies of KO and WT mice). We did, however, observe rectal prolapses in the breeding colony of our TNX KO mice population (shown in Fig. 1b), albeit at a low frequency (<1%). We did not observe rectal prolapses in our WT mice population.
Table 2Pregnancy in TNX-deficient miceGenito-urinary complicationsTNX KO miceWT miceNumber of miceNumber of miceMaternal death during pregnancy10Miscarriage20Obstruction during partus13Excessive bleeding of vagina (as seen at vaginal plug check)20Cannibalism after partus11Total number of evaluated pregnancies3631Fig. 1TNX KO mice abnormalities. Vaginal plugs, which are present after mating, are much more difficult to asses in TNX KO mice than in WT mice. This resulted in a significant increase of pregnancies in which the date of impregnation was unknown (a, *P<0.025, Chi-square test). Total evaluations: TNX KO mice, n=32; WT mice, n=28. In this study, no uterine prolapses were observed, although some TNX KO mice suffered from a rectal prolapse (b). Rectal prolapses were also observed in TNX-deficient patients, although the incidence in TNX KO mouse was lower (<1% in mice)
Structure of mouse uterus
The mouse uterus consists of three layers: an outer layer, which includes the single-cell layered perimetrium and a thin layer of connective tissue; a muscular layer, the myometrium, composed of two oppositely orientated layers of muscle; and an endometrium, which undergoes remodeling during the menstrual cycle and pregnancy. The endometrium contains luminal and glandular epithelial cells, stromal cells, resident immune cells of various types, and endothelial cells. No differences in the overall morphology of the uterine tissues were noticeable between TNX KO and WT mice (Fig. 2; uteri on 1 day postpartum) as assessed by light microscopy of H&E-stained sections. As we mainly focused on the ECM compartment of the uterus, we did not attempt to make quantitative analyses of the cell type distribution.
Fig. 2Structure of TNX KO and WT uterus. H&E-stained sections of the uterus of WT mice (a) and of TNX KO mice (b). No differences in the structure of the uteri were noticeable between TNX KO and WT mice. The mouse uterus consists of a thin outer layer, the perimetrium (P). A muscle layer, the myometrium (M), consists of two oppositely orientated layers of muscle, although this is difficult to distinguish in H&E-stained slides (E endometrium, L lumen). Bars 0.01 mm
TNX distribution in mouse uterus
TNX was present throughout the uterus of virgin mice and in the uterus during and after pregnancy (Fig. 3a-c). Immunostaining for TNX was discontinuous in the perimetrium (Fig. 3a-c,f). TNX was detected in the endometrium, although the epithelium lining the endometrium was negative for TNX (Fig. 3d). Furthermore, TNX was present in the layers of connective tissue ensheathing the muscle bundles of the myometrium (Fig. 3f). Overall, no change in the localization of TNX immunostaining was observed during and after pregnancy. The specificity of our TNX antibody was demonstrated by the complete absence of positive staining in the tissues of the TNX KO mice (Fig. 3e,g).
Fig. 3Immunostaining of TNX in the uterus (P perimetrium, M myometrium showing longitudinal muscle bundles, M* myometrium showing transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (green) is present throughout the uterus of virgin mice (a) and in the uterus during and after pregnancy, shown for uteri at 13 days pregnant (b) and 3 weeks postpartum (c). Cell nuclei are stained with DAPI (blue). TNX is present in the endometrium (d) and the layers of connective tissue ensheathing muscle bundles of the myometrium (f). TNX immunostaining of the perimetrium is relatively weak (a-c, f). The epithelium of the lumen is negative for TNX (d). The specificity of the TNX antibody is demonstrated in e, g. d–g Uteri at 3 weeks postpartum. Bars 50 μm
Collagen distribution in mouse uterus
We have previously demonstrated the binding and the colocalization of TNX with collagen types I, II and V, in the skin (Egging et al. 2006a, b). In the uterus, TNX also colocalizes with major fibrillar collagens types I, III, and V, as is shown for uteri 3 weeks postpartum (Fig. 4). The collagens are present in the perimetrium, the endometrium, and the layers of the connective tissue ensheathing the muscle bundles of the myometrium. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are found between WT and TNX KO mice (data not shown), which is in agreement with our observations in skin (Egging et al. 2006b). TNX does not exclusively colocalize with collagens, however, because TNX also cololalizes with other ECM components such as (tropo)elastin. Collagen type XII, an interaction partner of TNX (Veit et al. 2006), is present throughout the uterus (Fig. 5). Collagen type XII occurs in the transverse muscle bundles of the myometrium around blood vessels and around the lumen, although the signal intensity is not completely continuous around the lumen and blood vessels. TNX and collagen type XII are both present throughout the uterus. No differences in the collagen type XII localization in the uterus between WT (Fig. 5a,c,e,g) and TNX KO (Fig. 5b,d,f,h) mice have been observed.
Fig. 4TNX colocalizes with major fibrillar collagens types I, III, and V (P perimetrium, M myometrium with longitudinal muscle bundles, M* myometrium with transverse muscle bundles, E endometrium, Epi epithelium of the lumen, L lumen). TNX (b, e, h, green) and collagen types I (a), III (d), and V (g, red) colocalize (c, f, i, yellow, orange, magenta) in uteri 3 weeks postpartum. Similar results have been obtained for uteri from virgin, 13-days pregnant, and 1-day postpartum mice. No differences in collagen type I, III, and V immunostaining are apparent between WT and TNX KO mice (data not shown). In k, l, m, higher magnification images of the myometrium are shown to demonstrate the colocalization of the fibrillar collagens with TNX (collagen type III is used as an example). Bars 50 μmFig. 5Immunostaining of collagen type XII in uterus (P perimetrium, M myometrium with longitudinal muscle bundles, E endometrium). Collagen XII (red) is present throughout the uterus. Immunostaining appears to be the strongest in the transverse muscle bundles of the myometrium (M*), around blood vessels (BV), and around the lumen (L), although the signal intensity is not always completely continuous. No differences in the collagen type XII localization in the uterus between WT (a, c, e, g) and TNX KO (b, d, f, h) mice are observed. Bars 50 μm
Elastic fibers in mouse tissues
Elastic fibers in the skin of TNX-deficient patients are known to be abnormally shaped (Zweers et al. 2004), and thus we have investigated their structure in TNX KO mouse tissues. Elastic fibers in the uterus are mostly located in the myometrium and perimetrium (Fig. 6a,b). The endometrium contains few elastic fibers as shown for WT (Fig. 6a) and TNX KO (Fig. 6b) mice. In the myometrium and perimetrium, positive staining for elastin is seen predominantly in the layers of connective tissue ensheathing the transverse muscle bundles (Fig. 6c,d). Despite the relative low abundance of mature elastic fibers in the endometrium, the presence of elastin can be detected by immunohistochemistry, possibly a consequence of the sensitivity of the detection methods (Fig. 6c-d). Elastin localization is similar for WT (Fig. 6c) and TNX KO (Fig. 6d) mice. We have previously demonstrated binding of TNX with elastin and the colocalization of these two proteins in skin (Egging et al. 2006a, b). Consistent with these findings, elastin also (partially) colocalizes with TNX in the uterus, as shown in the myometrium (Fig. 6e). Colocalization of TNX with elastin is not complete, since TNX also colocalizes with fibrillar collagens; however, all elastin-reactive material colocalizes with TNX. In previous work, we have showed an increase in elastin-positive material in the skin of aging TNX KO mice (Egging et al. 2006a, b). Although, by light microscopy, the elastic fibers in the TNX KO mouse skin do not appear to be abnormally shaped, an increase in staining might have resulted from an increase in less mature or less organized elastin fibers, an increase in soluble tropoelastin within the matrix, and/or the consequence of more immunoreactive epitopes being available because of an increased susceptibility for degradation. These results are consistent with our past observations of elastic fibers in the skin of TNX-deficient patients in whom irregular and immature elastic fibers and fibers devoid of microfibrils can be seen at the ultrastructural level (Egging et al. 2006b; Zweers et al. 2004). Thus, to expand these findings further in skin and to investigate the elastic fibers in the uterus, we have performed an ultrastructural analysis of the elastic fibers in several tissues. Elastic fibers in WT (Fig. 7a) and TNX KO mice (Fig. 7b) uteri do not appear to differ in shape or size (shown for uterus 3 weeks postpartum). Similarly, in aorta, the elastic laminae of WT (Fig. 7c) and TNX KO mice (Fig. 7d) are similar in shape and number (aorta of 9-month-old mice). In skin of older TNX KO mice (as shown for 9-month-old mice), however, a difference in elastic fiber ultrastructure is apparent compared with WT skin. In addition to normal-appearing elastic fibers, irregular elastin aggregates, as seen in the skin of TNX-deficient patients (Zweers et al. 2004), can be observed (Fig. 7e,f). These elastin aggregates are not found in the skin of 2-month-old TNX KO mice or in 9-month-old WT mice. Interestingly, elastic fibers in the skin of 9-month-old TNX KO mice often appear larger than those of WT mice of the same age (compare Fig. 7g,h).
Fig. 6Elastin and elastic fibers in the uterus (L  lumen). Elastic fibers (purple, modified Hart’s staining) are mostly located in the myometrium (M, M*) and perimetrium (P), whereas the endometrium (E) appears to contain fewer elastic fibers in WT (a) and TNX KO (b) mice. No elastic fiber abnormalities are found in the TNX KO mice. Elastin immunostaining (red) is observed in the myometrium, predominantly in the transverse bundles (M* in c, d). The layers of connective tissue ensheathing the muscle bundles of the myometrium (M) and perimetrium (P) are stained positively for elastin (red). Strong elastin staining is also seen in the endometrium (E). Elastin immunoreactivity is similar for WT (c) and TNX KO (d) mice. Elastin (red) colocalizes (orange) with TNX (green) in the myometrium (M* in e). Not all TNX colocalizes with elastin as TNX also colocalizes with different collagen types (Fig. 4). Bars 50 μm Fig. 7Ultrastructural evaluation of elastic fibers. Elastic fibers in WT (a) and TNX KO (b) mouse uterus do not appear to differ in shape or size. The dark structures (arrows) are elastic fibers (shown for uterus at 3 weeks postpartum). The elastic laminae (arrows) in the aorta of WT (c) and TNX KO (d) mice are similar in shape and number (aorta of 9-month-old mice). Skin of older TNX KO mice (9 months old) shows differences in elastic fibers from those of WT skin. Irregular elastin aggregates can be observed in the TNX KO mouse skin (e, near a sebaceous gland). A higher magnification of an elastin aggregate (arrow) is shown in f. These aggregates were not found in skin of 2-month-old TNX KO mice or in 9-month-old WT mice. No irregularities in the shape of elastic fibers are observed in 9-month-old TNX KO mice skin; however, larger elastic fibers than in WT mice (g) are often observed in the TNX KO mice (h, arrows elastic fibers). Bars 0.5 μm (a, b), 10 μm (c, d), 2 μm (e, g, h), 1 μm (f)
Discussion
GU complications occur frequently in patients with various types of EDS (Carley and Schaffer 2000; Ramos-e-Silva et al. 2006; Lind and Wallenburg 2002; Kuczkowski 2005; Parry and Strauss 1998; Pepin et al. 2000). In classical and hypermobility types of EDS, which are the most common, the outcome of pregnancy is generally favorable. Maternal complications however, such as postpartum hemorrhage and pelvic instability, are more common than in the general population (Ramos-e-Silva et al. 2006; Kuczkowski 2005; Parry and Strauss 1998). Furthermore, EDS patients appear to have an elevated risk of uterine prolapse (Carley and Schaffer 2000; Lind and Wallenburg 2002). Pregnancy in patients with vascular type EDS may even lead to maternal death through uterus or vessel rupture (Pepin et al. 2000). We have investigated pregnancies and GU abnormalities in TNX-deficient woman of reproductive age. Generally, pregnancy is without major complications in TNX-deficient patients, apart from one noted incident of postpartum hemorrhage. However, uterine and vaginal prolapse regularly occur in TNX-deficient women, even at a young age, suggesting laxity of GU tissues. Premature rupture of fetal membranes is a risk in pregnancy with EDS affected fetuses (Lind and Wallenburg 2002; Parry and Strauss 1998). No premature births have been observed in the offspring of the TNX-deficient patients; however, some TNX-deficient patients had been born prematurely. Obviously, some caution must be taken in making conclusions and extrapolating data from such a small group of TNX-deficient woman.
Complete TNX deficiency in humans is a rare condition, and so far, only a few patients have been identified. We have therefore investigated pregnancy and uterine tissue structure in our TNX KO mouse model. No gross significant differences have been found with regard to abnormalities during pregnancy or reproduction between the TNX KO and WT mice. A trend toward a reduction in the length of pregnancy in TNX KO mice. The only significant difference has been observed in the location of the vaginal plugs, which suggests laxity in the vaginal wall.
In a previous study, we have not found any differences in cutaneous collagen deposition between TNX KO and WT mice (Egging et al. 2006b), as has been observed in the skin of TNX-deficient patients (Schalkwijk et al. 2001; Zweers et al. 2004). Alterations in collagen deposition in the skin of TNX KO mice is, however, a matter of debate (Egging et al. 2006b; Mao et al. 2002; Minamitani et al. 2004). In the present study, we have found elastin aggregates and enlarged elastic fibers in 9-month-old skin of TNX KO mice at the ultrastructural level; this is in accordance with our previously published data concerning increased elastin staining in aging TNX KO mouse skin (Egging et al. 2006b). We have detected no differences in elastic fibers in the uterus or elastic laminae of the abdominal aorta between TNX KO and WT mice suggesting a specific role for TNX in the maturation or maintenance of elastic fibers in skin. However, abnormal elastic fibers might not be present in the uterus, since the ECM is constantly being remodeled during the menstrual cycle and pregnancy.
Although the TNX KO mice appear to have a much milder phenotype compared with TNX-deficient humans, the skin of TNX KO mice is significantly weaker and more lax compared with WT mice (Bristow et al. 2005; Burch et al. 1997; Egging et al. 2006b; Lindor and Bristow 2005; Mao et al. 2002; Matsumoto et al. 2001; Minamitani et al. 2004; Schalkwijk et al. 2001; Zweers et al. 2004). Our data suggest a more lax vaginal canal in TNX-deficient patients and to a certain degree in TNX KO mice. An attractive speculation, considering the observations from our studies in mice, is that TNX is essential for tissue integrity, independent of the quantity of collagen deposition or elastic fiber structure. In conclusion, we would advise obstetricians and gynecologists to monitor TNX-deficient patients closely, as they are likely to have weaker GU connective tissue and could therefore be at risk of GU complications.

tenascin-x
human
ehlers-danlos syndrome
pregnancy
collagen
elastin
mouse (tnx knockout; c57bl/6)


Feasibility study of computed tomography colonography using limited bowel preparation at normal and low-dose levels study

The purpose was to evaluate low-dose CT colonography without cathartic cleansing in terms of image quality, polyp visualization and patient acceptance. Sixty-one patients scheduled for colonoscopy started a low-fiber diet, lactulose and amidotrizoic-acid for fecal tagging 2 days prior to the CT scan (standard dose, 5.8–8.2 mSv). The original raw data of 51 patients were modified and reconstructed at simulated 2.3 and 0.7 mSv levels. Two observers evaluated the standard dose scan regarding image quality and polyps. A third evaluated the presence of polyps at all three mSv levels in a blinded prospective way. All observers were blinded to the reference standard: colonoscopy. At three times patients were given questionnaires relating to their experiences and preference. Image quality was sufficient in all patients, but significantly lower in the cecum, sigmoid and rectum. The two observers correctly identified respectively 10/15 (67%) and 9/15 (60%) polyps ≥10 mm, with 5 and 8 false-positive lesions (standard dose scan). Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value (polyps ≥10 mm). Eighty percent of patients preferred CT colonography and 13% preferred colonoscopy (P<0.001). CT colonography without cleansing is preferred to colonoscopy and shows sufficient image quality and moderate sensitivity, without impaired diagnostic value at dose-levels as low as 0.7 mSv.
Introduction
Since computed tomography colonography (CTC) has shown good results in detecting clinically relevant polyps [1–6], studies have been performed focusing on increasing patient acceptance by reducing the bowel preparation. This can be done by adding an oral contrast agent to meals (fecal tagging) [7–12] in combination with a low-fiber diet, and sometimes lactulose for stool softening, thereby obviating extensive cleansing.
Different studies have been performed using barium, iodine or a combination of both as tagging material. However, only a limited number of these studies on CTC have used limited bowel preparation. Few have evaluated the diagnostic value and patient acceptance of CTC. In parallel with the development of limited bowel preparation strategies, (ultra) low-dose protocols have been evaluated [13–15].
To our knowledge, to date no studies have evaluated the effect of dose reduction on sensitivity and specificity in patients with a limited bowel preparation. Only one study [7] has evaluated limited bowel preparation at a relatively low dose level (140 kVp, 10 mAs). This study has shown very good results; however, these data have not been reproduced yet. Moreover, as only one dose-level was studied, the effect of dose reduction remains unknown.
The purpose of this study was to evaluate limited bowel-preparation CTC using an oral contrast agent (amidotrizoic acid) in terms of image quality, patient acceptance and polyp visualization using conventional colonoscopy (CC) as a reference standard. A second objective was to determine the effect of substantially reducing the radiation dose levels on the diagnostic accuracy of limited bowel preparation CTC, again using CC as the reference standard.
Materials and methods
Study population
Patients of the Onze Lieve Vrouwe Gasthuis at increased risk for colorectal cancer (personal or family history of colorectal polyps or cancer) [16] who were scheduled to undergo CC between April 2002 and August 2003 were invited to participate in the study. Exclusion criteria were: impossibility to understand the information/informed consent, age below 18 years, pregnancy and inflammatory bowel disease. The study was approved by the institutional review board of the Onze Lieve Vrouwe Gasthuis and Academic Medical Center. All patients gave written informed consent.
CTC bowel preparation and scanning protocol
Four weeks prior to the CC patients were asked to ingest amidotrizoic acid (20 mg/ml, made by the hospital pharmacy, 11.7 mg I/ml; corresponding to approximately 30 times diluted Gastrografin370®) three times a day (100 ml at breakfast and lunch, 300 ml at dinner) with a low-fiber diet (well cooked vegetables and meat, no fibrous fruit, no whole-wheat products, no nuts) starting 2 days prior to CTC. Lactulose (12 g, lactulose CF powder 6 g/sachet, Centrafarm, Etten-Leur, The Netherlands) was taken in the morning for 3 days prior to CTC for stool softening. Twenty mg of butylscopolaminebromide (Buscopan; Boehringer-Ingelheim, Ingelheim, Germany), when contraindicated, 1 mg of glucagon hydrochloride (Glucagen; Novo-Nordisk, Bagsvaerd, Denmark) was administered intravenously. The colon was insufflated with a CO2-air mixture (13.2% vol.) using a flexible catheter until patients experienced discomfort (±2–3 l). Patients were scanned in prone and supine position with a four-slice MX8000 (Philips Medical Systems, Best, The Netherlands) CT scanner (120 kV, rotation time 0.75 s, pitch 1.25, collimation 4*2.5 mm, section thickness 3.2 mm, and reconstruction interval 1.6 mm, 50 or 70 milliampere-second (mAs); 70 mAs if the abdominal circumference was >102.5 cm, scan time 20–25 s).
Colonoscopy
Prior to CC, each patient was instructed to ingest 4 l of a macrogol solution (Colofort macrogol 4000 sachets, Ipsen, Hoofddorp, The Netherlands), starting on the evening prior to the CC. CC was performed by one of three experienced gastroenterologists of the Onze Lieve Vrouwe Gasthuis (experience as gastroenterologist: 23, 12 and 4 years) and recorded on videotape. Patients received sedatives [5 mg midazolam (Dormicum; Roche, Basel, Switzerland)] and analgesics [0.05 mg fentanyl (Janssen Pharmaceuticals, Beerse, Belgium)] at the request of the patient. If polyps were present, the location was determined and size was estimated, based on comparison with an open biopsy forceps prior to removal. A polyp was considered flat if its height was less than one-half of the diameter of the lesion [17]. Only segments visualized at CC were used for analysis.
Simulation of low-dose CT colonography
To study polyp detection at lower dose, CTC examinations were simulated with a lower dose/mAs-value using an established simulation technique [18, 19] that has been applied in two earlier CTC studies [13, 14]. This simulation method consists of the controlled increase of noise in the raw CT transmission data prior to reconstruction of the images. In this way, scans were simulated at 20 mAs (in the order of 2.3 mSv for two scans: supine and prone) and 6 mAs (±0.7 mSv). A previous simulation study has demonstrated that the radiation dose of CTC after extensive bowel cleansing can be reduced down to 0.2 mSv [13]. Since the contrast between tissue and tagged fecal material in limited bowel preparation colons is lower than that between air and tissue, we chose 0.7 mSv as the lowest simulated dose-level next to 2.3 mSv.
CTC evaluation
An unblinded research fellow (J.F., experience: evaluated 300 CTC) matched findings at CC (polyps ≥6 mm and colorectal cancer) with the CTC data to provide a frame of reference for true positive findings at CTC. Only exact matches in shape, size and location were scored as a positive match. All images were then independently evaluated using a primary 2D display mode by three blinded observers. The first observer (research fellow, R.v.G., experience: compared >300 CTCs with CC videos) evaluated the original CTC scan for image quality and for polyps. Scores were assigned (per patient/segment, Table 1) for overall image quality, luminal distension, homogeneity and presence of stool. The second observer evaluated the original CTC for polyps and marked those examinations that were not diagnostic. If both observers rated a CTC as not diagnostic, this patient was excluded from analysis. The third observer first evaluated the simulated 0.7 mSv scans for presence of polyps at least 4 weeks after the simulated 2.3 mSv scans, and again at least 4 weeks later the original CTC scans. All lesions were measured and screenshots were taken. The second observer (V.v.d.H., experience: 8 years of clinical CT experience as abdominal radiologist) and the third observer (A.v.R., a research fellow in radiology with no prior experience with CTC) had had a learning curve in evaluating 50 CTCs with CC feedback.
Table 1Scales used by observer 1 to rate image quality (upper part) and scales used by the patients to rate experience and preferenceObserver 1:ScaleImage quality (patient, segment)*1: poor, not diagnostic; 2: moderate, diagnostic with limitations; 3: good, diagnostic with minor limitations; 4: excellent, no limitationsDistension (segment)1: collapsed; 2: poorly distended; 3: only moderately distended but segment is distended over its full length; 4: good; 5: very goodHomogeneity (segment)1: poor; 2: moderate; 3: good; 4: very goodPresence of stool (segmemt)1: large amount of stool, segment fully filled; 2: moderate amount of stool, ~50% of lumen filled; 3: small amount of stool; 4: only contrast layer on the wall; 5: no stool at allPatients:Most burdensome aspectCTC preparation: diet, lactulose, contrast agentCTC: iv puncture, catheter placing, insufflation, breathholds, prone positionCC: iv puncture, moving of scope, air insufflation, monitoring after CCHow burdensome/painfulNot, mild, moderate, severe, extremePreferenceDefinitely CTC, probably CTC, possibly CTC, indifferent, possibly CC, probably CC, definitely CCMost reluctant factorCC, bowel preparation prior to the CC, CTC, the limited bowel preparation prior to the CTC*The items were scored per patient and per segment.
Polyp detection and image quality
A polyp detected at CTC was labeled as true positive if three criteria were met: segmental location and location within the segment corresponded with CC (when situated near the borders of the segment, localization in the adjacent segment was also accepted), the polyp size as estimated by the endoscopist (open forceps) corresponded with size as measured on CTC (50% margin based on the CC size was allowed), and appearance (morphology) closely resembled that of the corresponding polyp at videotaped CC. The unblinded research fellow determined the nature of false-positive findings ≥10 mm by reviewing the videotaped CC and CTC. If the polyp was possibly missed at CC, a repeat CC was called for.
Patient questionnaires
Patient experience and preference were evaluated by six questionnaires (scales are shown in Table 1): 2 weeks prior to the CTC, directly after the CTC bowel preparation, after the CTC, after the CC preparation, after the CC and 5 weeks later. The first and the last questionnaires were sent by mail. After the CTC bowel preparation, patients were asked how burdensome they rated the CTC bowel preparation, and the most burdensome aspect. After CTC they rated how much pain they had experienced and how burdensome the CTC had been. Prior to CC patients were asked how burdensome the extensive bowel preparation had been. After the CC they were asked how much pain they had experienced and how burdensome the CC had been. After the CC and 5 weeks later, patients were asked for their preference for either CTC or CC assuming that both techniques were equally accurate, while in 20% of CTC examinations clinically relevant lesions would be shown, necessitating a therapeutic CC. Two weeks prior to the CTC, directly after the CC and 5 weeks later, patients were asked what they were most reluctant to undergo.
Statistical analysis
Differences in quality between CTC images of different segments were assessed using ordinal regression analysis. For this analysis we first determined the best segment and used this segment as reference segment. The regression coefficient of each segment estimates the change in the log transformed diagnostic odds ratio (DOR) compared to the reference segment. It can be interpreted, after antilogarithm transformation as relative diagnostic odds ratio (RDOR). A lower RDOR implies inferior image quality of the respective segment compared to the reference segment. Differences in polyp detection rates between CC and lower dose CTC were assessed with the McNemar test for paired proportions. The Wilcoxon signed rank test statistic was used to evaluate differences in patient experience between CTC and CC and to evaluate differences in preference between the questionnaire filled out directly after the procedures and 5 weeks later. The data were first dichotomized as preference for CTC versus preference for CC. These differences were tested using the chi-square test statistic. All P-values <0.05 were considered to indicate statistical significance.
Results
Sixty-three eligible patients (53%) gave written informed consent. Two patients were excluded. One patient was excluded since he did not undergo CC for unknown reasons. In another patient with a 20-mm carcinoma opposite the ileocecal valve at CC, the CTC was rated as of no diagnostic value by both observers (parts of the large bowel not in the field of view, stool not tagged sufficiently, and breathing movement artifacts). Therefore, 61 patients were analyzed (Table 2); 51 of these 61 patients could be reviewed regarding dose reduction.
Table 2Baseline characteristics of the study populationIncluded61Male/female40/21Age in years: mean ± sd† (range)61 ± 12 (27–81)Indication:H/O† colorectal polyps38H/O colorectal carcinoma9F/H† of colorectal polyps or cancer14Coexistent complains: Abdominal pain/hematochezia/altered bowel habits12/4/3Colonoscopy: number of polyps/patients with polyps:Any size94/38≥6 mm28/20≥10 mm15/12≥10 mm initial colonoscopy13/11Morphology of polyps ≥6 mm (sessile/stalked/flat/CRC)12/7/7/2Morphology of polyps ≥10 mm (sessile/stalked/flat/CRC)3/6/4/2Colonoscopy: no. of patients receiving Sed+analg/sed/analg/none29/7/3/22Stool consistency prior to CTC (diarrhea/soft/normal)15/13/33Abdominal pain prior to CTC (major/minor)1/7Flatulence prior to CTC (major/minor)3/27Spasmolitycs during CTC (Buscopan/Glucagon/neither)47/12/2†sd: standard deviation; H/O: history of; F/H: family history, sed: sedatives. Analg: analgesics
In five patients it was not possible to fully inspect the colon endoscopically. In one patient a 14-mm polyp was seen at CTC in a segment not inspected at CC and confirmed at surgery (not included in the analysis). In two patients, repeat CC showed a 30-mm polyp and a 30-mm carcinoma (included in the analyses), both missed at the initial CC.
Image quality
Overall image quality was rated as excellent in 2 patients, good in 41 and moderate in 18. One patient who received a score of poor/not diagnostic was excluded. Image quality was significantly lower (Fig. 1) in the cecum (both positions), sigmoid (prone position) and rectum (supine position). This was mainly due to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum.
Fig. 1Figure showing RDOR with confidence intervals of all six segments of the colorectum in both prone and supine position regarding overall image quality, distension, presence of stool and homogeneity. All segments are compared to the best segment (DOR by definition 1). Confidence intervals not reaching 1 indicate significantly inferior results
Diagnostic value
Including the findings at repeat CC, 12 patients had 15 polyps ≥10 mm (including 2 carcinomas of 35 mm and 30 mm) while 20 patients had in total 28 polyps ≥6 mm (Fig. 2). Twenty-three of 61 patients did not have any polyps at colonoscopy. The initial CC detected 13 of the 15 large polyps (sensitivity 87%). The per-patient sensitivity for the initial CC was 92% (11/12). The unblinded research fellow was able to match 13/15 (87%) large polyps (in 10/12 patients: 83%), while 2 large flat lesions were not visible in retrospect. For polyps ≥6 mm he matched 23/28 (87%) polyps (3 additional flat lesions not visible in a patient with already a large flat lesion) in 18/20 (90%) patients. Observers 1 and 2 correctly identified 10/15 (67%) and 9/15 (60%) polyps ≥10 mm, with respectively 5 and 8 false-positive lesions (Table 3). All but two were explained as stool.
Fig. 2A 76-year-old woman with a 7-mm stalked polyp (arrow) submerged in a layer of tagged stool at CTC (original dose; a: supine position, b prone position) and at colonoscopy (c)Table 3Performance characteristics per observer per size categoryVariablePolyps ≥10 mmPolyps ≥6 mmObserver:Observer:1212Analysis according to polypSensitivity10/15 (67%)9/15 (60%)17/28 (61%)15/28 (54%)FP582028PPV10/16 (63%)9/17 (53%)17/38 (45%)15/43 (35%)Analysis according to patientSensitivity8/12 (67%)8/12 (67%)13/20 (65%)13/20 (65%)Specificity45/49 (92%)41/49 (84%)30/41 (73%)25/41 (73%)PPV8/12 (67%)8/16 (50%)13/24 (54%)13/29 (45%)NPV45/49 (92%)41/45 (91%)30/37 (81%)25/32 (78%)PPV: positive predictive value; NPV: negative predictive value; FP: false positives; CI: confidence interval
Polyp detection at lower dose-levels
In 51 of 61 patients, we were able to simulate and reconstruct data at lower dose levels. In 10 of 61 patients this was not possible due to missing original raw data. Dose reduction down to 0.7 mSv was not associated with significant changes in diagnostic value for patients with lesions ≥10 mm (Table 4, Figs. 3 and 4). Only focusing on the data of these 51 patients, reader 3 showed diagnostic values comparable to or even better than reviewer 1 and 2: sensitivity for reviewer 1 and 2 both 5 out of 10 large polyps with respectively 5 and 8 false-positive lesions ≥10 mm and respectively 10 and 9 out of 20 polyps ≥6 mm with respectively 20 and 25 false-positives ≥6 mm. Specificity for patients without lesions ≥6 mm was significantly lower (P=0.003) at 6 mAs when compared to the original dose.
Table 4Performance characteristics of observer 3 per dose levelVariablePolyps ≥10 mmPolyps ≥6 mmDose levelDose levelOriginal2.3 mSv0.7 mSvOriginal2.3 mSv0.7 mSvAnalysis according to polypSensitivity5/10 (50%)7/10 (70%)4 /10 (40%)11/20 (55%)13/20 (65%)10/20 (50%)FP22451120Analysis according to patientSensitivity5/9 (56%)7/9 (78%)4 /9 (44%)10/15 (67%)12/15 (60%)9/15 (45%)Specificity40/42 (95%)40/42 (95%)39/42 (93%)33/36 (92%)26/36 (72%)23/36SS (64%)SS: statistically significant. FP: false positivesStatistical analysis (only performed on the per patient data) showed that only the specificity for patients without lesions ≥6 mm was significantly lower (P = 0.003) at 0.7 mSv when compared to the original doseFig. 3Figure showing a large 12-mm false-positive lesion (arrow) in the transverse colon at three different dose levels in a 65-year-old male patient (a: 0.7 mSv, b: 2.3 mSv, c: original). At lower dose levels it is more difficult to differentiate this lesion from a true-positive lesion (polyp) when fecal material is insufficiently tagged, this especially applied to smaller lesions. Well-tagged material is clearly differentiated from colon wall, even at 0.7 mSv (arrowhead)Fig. 4Figure showing a large 10-mm stalked polyp (arrowhead) in the sigmoid at three different dose levels in a 57-year-old male patient (a: 0.7 mSv, b: 2.3 mSv, c: original)
Patient experience and preference
The 6 questionnaires were filled out by respectively 58 (95%), 61 (100%), 61 (100%), 56 (92%), 57 (93%) and 57 (93%) patients. Two weeks prior to the CTC patients were most reluctant to undergo CC, then the CC bowel preparation, thirdly the limited CTC bowel preparation. The CTC was feared least. After both procedures (fifth questionnaire), the order was the same. Five weeks later the bowel preparation prior to the CC was regarded to be the most burdensome, followed by the CC, then the CTC preparation and the CTC. None of the three aspects of the bowel preparation for CTC (the diet, lactulose and contrast agent) was rated significantly worse than the others (Fig. 5). Patients rated the limited bowel preparation (prior to CTC) less burdensome than the bowel preparation prior to CC (Fig. 6, P<0.001). Patients also experienced more pain during CC than during CTC (Fig. 6, P<0.001). The most burdening aspect of the CTC was insufflation of air (58%). The most burdening aspect of CC was the movement of the scope (59%). Directly after the CC (fifth questionnaire) 80% (43/54) of the patients preferred CTC for their next examination, 13% (7) preferred CC (P<0.001) and 7% (4) were indifferent (Fig. 6). Five weeks after the procedures, 71% (39/55) preferred CTC, 13% (7) preferred CC (P<0.001) and 18% (10) were indifferent. After 5 weeks there was no change (P=0.12) in preference of CTC as the next screening test.
Fig. 5Graph showing how patients rated the three different components of CTC with limited bowel preparation. How burdensome were the diet, lactulose and the contrast agent?Fig. 6Graphs show patients’ experience of the bowel preparation (upper left) and the CTC examination (upper right) and patient preference for one of the two modalities (CTC with limited bowel preparation versus CTC with standard bowel preparation, lower). How burdensome was the limited bowel preparation prior to CTC (grey) as compared to cleansing prior to CC (black)? How burdensome were the CTC (grey) and CC (black) examinations (upper right graphs)? What did participants prefer for their next examination (lower graph) directly after both examinations and in the questionnaire sent at home 5 weeks later?
Discussion
This study demonstrates that CT colonography without cleansing is preferred to colonoscopy and shows moderate sensitivity (60–67%) for polyps ≥10 mm without impaired diagnostic value at mSv levels as low as 0.7 mSv.
Image quality was good on average. Nevertheless, the cecum, sigmoid and rectum showed overall reduced image quality. Although this can be caused by the pelvis causing more noise, reduced imaged quality must probably be attributed to inferior homogeneity in the cecum and rectum and inferior distension in the sigmoid and rectum in the prone position. In contrast to the known problems in CTC with distension, which are solved by dual positioning, inferior homogeneity in the cecum and rectum are typical for the limited bowel preparation protocol. Insufficient homogeneity in the cecum is probably caused by the fact that patients ate food after the last amount of contrast agent was taken, resulting in inadequately tagged stool. The inhomogeneous stool in the rectum probably was caused by the fact that in patients with a long transit time stool was already shaped before the fecal tagging was started.
We assume that two adaptations are necessary to improve image quality. First, contrast must be taken as long as the patients are eating. Second, stool should be made softer, for example by replacing lactulose by a stronger osmotic laxative (e.g., low-dose magnesium salts), thereby also reducing the transit time and reducing the amount of non-tagged stool in the rectum. Although these adaptations increase the burden of the bowel preparation, they are slight and are required to improve image quality and thereby maybe the sensitivity and specificity.
Our study showed an average sensitivity of 63% for large polyps (with a limited number of large false-positive lesions). This is lower than the 100% average sensitivity described by Iannaccone et al. [7] using a more or less comparable bowel preparation; no fiber-rich food and 200 ml of 370 mg/ml iodinated contrast agent in 48 h (which is 6.3 times the amount given in our study), but no lactulose. In addition to the differences in bowel preparation, differences in experience of the readers or reader performance, difference in study groups (polyp prevalence and spectrum) might explain these discrepant results. A recent study [20] has shown inferior results regarding diagnostic value of CTC in populations at increased risk, as is used in the current study, possibly due to a relatively high number of hard-to-see polyps. When comparing our study with three large studies on CTC with extensive bowel preparation [21–23] (showing per polyp sensitivities of respectively 52%, 32–73% and 53% for polyps ≥10 mm), our results are comparable or slightly better.
Since two of five (observer 1) and two of six (observer 2) missed polyps were not seen in retrospect by the unblinded observer, the problem of missing large polyps can be regarded as predominantly an interpretation problem rather than a visualization problem. Future developments, such as better homogeneity, better distension, better learning curve, intuitive display modes, electronic cleansing and computer-aided detection can help in reducing the number of missed polyps.
Sensitivity and specificity were impaired at lower dose levels for patients with lesions ≥10 mm. This is especially of interest in screening protocols. Regarding lesions ≥6 mm, specificity was significantly lower at the simulated 0.7 mSv low-dose scans. Increased noise levels made it impossible to see the tiny air bubbles inside stool. These results may seem to be in contrast with those in an earlier report on low radiation dose CTC [13] showing fewer false-positive lesions. However, these studies in patients with extensively cleansed colons used a three-dimensional display mode and filters to smooth the bowel wall. By using these smoothing filters, small artifacts and small amounts of residual stool were masked when evaluating the colon three dimensionally. Making stool softer and scanning with thinner slices possibly allows for better visualization of the air bubbles, thereby reducing the number of false positives.
Patient acceptance was found to be very good in the current study. Patient preference was convincingly in favor of CTC; 71% of patients preferred CTC with limited bowel preparation to CC for their next examination. Other studies have shown discrepant results on patient acceptance of CTC versus CC, some favoring CTC [24–27], others CC [28, 29]. In a study where patients first underwent extensive cleansing and consecutively CTC and CC [24] fewer patients (71% directly after the CC) favored CTC when compared to our study (80% directly after CC). This difference cannot be attributed to the bowel preparation because of a different study setup.
Several potential limitations must be considered. The number of patients included is relatively low, especially for determining the detection parameters at the 2.3 and 0.7 mSv levels.
In the current study no segmental unblinding during CC was performed. Although repeat CC showed that two large false-positive lesions were missed at the initial CC, segmental unblinding has the advantage of evaluating all false positives.
Since no electronic cleansing was used and many polyps were submerged, 3D evaluation of these lesions was not of additive value. However, 3D evaluation with electronic cleansing might have given better results, especially since most missed polyps were visible in retrospect.
Although the two readers that had no previous experience in CTC underwent a learning curve of 50 patients, some studies have shown that this may not be sufficient for optimal polyp detection [30, 31]. For CTC using limited bowel preparation, the learning curve is possibly even longer.
When filling out the questionnaires, sensitivity for CC and CTC were assumed equal. If patients knew that the sensitivity of CTC was lower than for CC, this most likely would have influenced the preference for CTC negatively.
In conclusion, these results show that CTC with the limited bowel preparation protocol used in this study is feasible, even when using doses as low as 0.7 mSv levels. Although sensitivity was not as high as previously reported, this is most likely not due to the limited bowel preparation, but to interpretation problems. Technical developments will probably increase the sensitivity, while minor adjustments in bowel preparation may reduce the number of false positives, especially when using low-dose protocols. Since patient acceptance was very good, this technique can be regarded as promising in screening patients of populations with a low prevalence of polyps.

ct colonography
colonoscopy
fecal tagging
colonic neoplasm


Instrumented fusion of thoracolumbar fracture with type I mineralized collagen matrix combined with autogenous bone marrow as a bone graft substitute: a four-case report

In order to avoid the morbidity from autogenous bone harvesting, bone graft substitutes are being used more frequently in spinal surgery. There is indirect radiological evidence that bone graft substitutes are efficacious in humans. The purpose of this four-case study was to visually, manually, and histologically assess the quality of a fusion mass produced by a collagen hydroxyapatite scaffold impregnated with autologous bone marrow aspirate for posterolateral fusion. Four patients sustained an acute thoracolumbar fracture and were treated by short posterior segment fusion using the AO fixateur interne. Autologous bone marrow (iliac crest) impregnated hydroxyapatite-collagen scaffold was laid on the decorticated posterior elements. Routine implant removal was performed after a mean of 15.3 months (12–20). During this second surgery, fusion mass was assessed visually and manually. A bone biopsy was sent for histological analysis of all four cases. Fusion was confirmed in all four patients intraoperatively and sagittal stress testing confirmed mechanical adequacy of the fusion mass. Three out of the four (cases 2–4) had their implants removed between 12 and 15 months after the index surgery. All their histological cuts showed evidence of newly formed bone and presence of active membranous and/or enchondral ossification foci. The last patient (case 1) underwent implant removal at 20 months and his histological cuts showed mature bone, but no active ossification foci. This four-case report suggests that the fusion mass produced by a mineralized collagen matrix graft soaked in aspirated bone marrow is histologically and mechanically adequate in a thoracolumbar fracture model. A larger patient series and/or randomized controlled studies are warranted to confirm these initial results.
Introduction
Effectiveness of a bone graft can be described as having three core properties: osteoinductivity, osteoconductivity and osteogenicity [25, 28]. Autogenous bone graft is the only biological structure simultaneously possessing all three properties. However, the long-term morbidity due to autogenous bone harvesting can be as high as 30%, although improved technique has probably lowered the incidence [1, 2, 10, 26]. Allograft bone is the primary alternative to autograft for a number of spinal fusion procedures. However, allograft bone is a poor posterior onlay graft with rates of fusion reported to be consistently lower than autograft except in cases of pediatric deformity [4, 12, 17, 18]. Therefore, there has been an increasing shift in the past decade to the use of bone graft substitutes for spinal fusion.
One commercially available bone graft substitute comprised a Type I mineralized collagen matrix (MCM), coated with hydroxyapatite (Healos® Bone Graft Substitute, DePuy Spine, Inc, Raynham, MA, USA). Kraiwattanapong et al. [21] reported a 0% fusion rate for posterolateral fusion with MCM and bone marrow in rabbits. However, in that study, bone marrow was harvested from the iliac crest which is not a rich source of osteoprogenitor cells in rabbits. Despite these unique findings, the efficacy of this bone graft substitute has been demonstrated in a previous rabbit study of posterolateral fusion preformed by Tay et al. [29] harvesting bone marrow from rabbit tibiae, with a fusion rate of 100%. Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 11, 28]. The effectiveness of this method has been further enhanced by the use of a three-dimensional scaffold which avoids wash out of osteogenic bone marrow cells by blood circulation. In humans, Kitchel has recently reported two lumbar posterolateral fusion studies [19, 20] demonstrating equivalent rates of fusion for MCM to autograft using CT scans for assessment.
The current understanding of fracture healing is primarily based on animal histological and radiological studies. The majority of human studies discussing fusion rates are based on radiological fusion criteria which are not completely reliable, even when CT scans are utilized [6]. In a 1993 study involving spinal implant removal following fusion, Blumenthal et al. [3] found an overall agreement between preoperative radiographs and surgical findings in only 69% of the cases. Brodsky et al. [5] found a similar overall agreement in their study published in 1991. The ultimate method to assess the quality of a fusion is perioperative manual palpation and histological analysis of a bone biopsy following implant removal.
There are no reports in the literature of the histological behavior of this bone graft substitute in humans. We present here our findings of radiological and histological results on four patients that had surgery performed for an acute thoracolumbar fracture without neurologic deficit.
Materials and methods
Five consecutive patients underwent surgical management for an acute vertebral fracture of the thoracolumbar spine without neurologic deficit. There were three females and two male patients, and the mean age was 47 years (range 30–64). Three patients sustained their fracture after a fall from a horse, one patient after a fall from a scaffold, the last after a fall from a tree. All patients were initially admitted to our institution, which is a level one trauma facility. Initial management was applied according to Advanced Trauma Life Support (ATLS®) guidelines. Four out of the five patients were diagnosed with no other lesion than the vertebral fracture. The patient who fell from a scaffold was also diagnosed with pulmonary contusions and severe tibial pilon fractures, open type IIIA according to Gustilo’s classification [13, 14]. All patients were worked-up under standard hospital protocol which included plain anteroposterior and lateral radiographs as well as a CT scan of the spine.
The AO classification was used to characterize the type of fracture in each patient [23]. Three patients sustained an L1 fracture: two patients had a type A2.3 (Burst-split), one patient had a type B1.2 fracture (flexion-distraction). One patient sustained a type C2.1 fracture (flexion-distraction with rotation) of T12. The fifth patient sustained a type A3.1 fracture of L2 (lateral burst).
All patients were conscious and given the choice between non-surgical and surgical management of their fracture after discussing with each of them the advantages and disadvantages of each treatment method, and alternatives. All patients signed an informed written consent to be included in this pilot study. All surgeries were performed by two fellowship trained surgeons (AAF and AJK).
Surgical technique
The short segment fusion-stabilization technique as described originally by Lindsey and Dick [9, 22] was utilized. The spine was approached posteriorly with a midline incision. Musculature was detached subperiosteally and the fracture was exposed posteriorly, both inferiorly and superiorly, to immediately adjacent vertebrae, taking care to preserve the facet joints of the intact segment. Following this, pedicle screws were placed in one vertebra above and one vertebra below the fracture under fluoroscopic control. Whenever possible, the authors attempted to stabilize (and fuse) only one segment if pedicle screws could be placed in the fractured vertebra, typically hemiburst fractures (AO classification type A3.1) where the inferior part of the vertebral body is primarily intact. This was not possible in either of the patients with type A fractures in this study. Following pedicle screw placement, careful decortication of the facet joints and posterior arch of the fractured vertebra was performed. Typically, transverse processes are not decorticated unless a laminectomy to achieve decompression is necessary.
Percutaneous aspiration of 10 cc of autogenous bone marrow from the posterior iliac crest was performed. Two 5 cc rectangles of the MCM graft were saturated with the bone marrow aspirate and laid bilaterally on the posterior elements previously decorticated. No other bone, bone graft extender, or substitute was placed as part of the graft. In the patient who sustained a lateral burst fracture, we performed a transpedicular bone grafting of the vertebral body, but no other graft substitute other than Healos® was used for the posterior fusion stabilization. Rods were connected to the pedicle screws and fracture reduction in the sagittal plane, and when necessary coronal plane, was achieved by means of ligamentotaxis through the “fixateur interne” (USS®: Universal Spine System, Synthes Spine, Paoli, PA, USA) as described by Walter Dick [9, 22].
Standard procedure with this instrumentation is to remove it at 12 months following surgery when radiological and clinical healing is evident. There are two major reasons for this: (1) implant removal allows the fixed but non-fused segment to recover some mobility which may protect the adjacent segment from accelerated degeneration; (2) in general, after the fracture is radiologically healed, patients experience some discomfort when trying to increase their physical activity. Patients clearly differentiate this discomfort from the pain they had from the fracture and during the healing period. The USS system is in fact a very bulky implant, which the authors believe, is eventually responsible for this discomfort.
Due to surgeon and patient availability, the time to instrumentation removal is often variable, but always performed at a minimum of 12 months following the index surgery at our institution. When radiological and clinical healing was achieved, implant removal was performed in four patients. The fifth patient declined implant removal following healing of the fracture because she did not experience any discomfort. Technically, the second surgery required a very short general anesthesia and lasted approximately 30 min from skin incision to skin suture.
During the second surgery, the mechanical stability of the fused segment was tested using thin curettes introduced in the pedicle screw tracts and manual stress was applied sagittally. A bone biopsy was performed in the heart of the fusion mass which was clearly distinguishable from the lamina and sent for histological analysis. Biopsies were sent in formalin to the institution’s pathology department. They were then decalcified in formic acid, embedded in paraffin and cut in 3-μm slices with a microtome. They were further colored with hematoxylin–eosin and analyzed under a microscope at magnifications ranging from 20× to 400×.
Radiological assessment
Anteroposterior and lateral radiographs were taken in the supine position immediately after surgery, in the standing position a few days after surgery, then at approximately 6 weeks, 3, 6, and 12 months following the index surgery, as well as following implant removal. Assessment of healing radiographically was based only on the remodeling of the vertebral body. Overall fusion assessment was based on radiographic healing of the vertebral body as well as patient pain, function, and activity level. Fusion of the posterior elements could not be assessed radiologically. CT scan was not used because it would not have been possible to assess fusion status of the posterior elements due to metallic artifacts from implants. When smoothening of bony edges and some sclerosis of the fractured vertebral body became visible, the fracture was considered to be healed. This was also based on the patient’s subjective appreciation. When fracture and surgery pain regressed and the patient felt he or she could go back to almost all previous all day activity, the fracture was considered clinically healed. No specific clinical outcome scale was used in this limited patient sample.
Results
The four patients who had the secondary surgery underwent implant removal after a mean period of 15.3 months from the index surgery (12–20). All patients had returned to their previous activity at latest follow-up, except one who was involved in worker’s compensation litigation.
During the second surgery, we visually confirmed that the resorbable matrix had been replaced by new bone (Fig. 1) and that this new bone had formed at the location where the MCM graft had been laid. Manual palpation and sagittal stress through curettes placed in the pedicle tracts demonstrated that no movement could be detected visually in any of the four patients.
Fig. 1This 30-year-old female sustained an AO type C2.1 fracture of T12 after a fall from a horse. Initial work-up in the ER did not show any other lesion. She was neurologically intact. We performed a T11-L1 posterior short segment fusion as previously described. Implant removal was performed 14 months following the index surgery. The biopsy demonstrated irregular bony trabeculae, foci of enchondral ossification and numerous osteoblasts, all characteristic elements of ongoing bone remodeling. Several aggregates of lymphoplasmocytes were observed and have been attributed to a foreign body inflammatory type of response. a Preoperative sagittal CT. b Preoperative lateral radiograph. c Lateral radiograph at 5 months postop. d Perioperative image of extensive new bone growth during the secondary surgery. e AP radiograph 15 months following the index surgery after implant removal. f Histology at 100× magnification. g Histology at 400× magnification
Histological analysis of these four cases showed clear evidence of newly formed bone. Patients 1, 3, and 4 had implant removal performed between 12 and 15 months follow-up. Their histological cuts also showed several foci of active membranous and/or enchondral ossification. Interestingly, patient 2 had implant removal performed at 20 months of follow-up and though his histology cuts showed newly formed bone, he had no visible active ossification focus. Patient 3 had implant removal performed at 14 months. His histology showed newly formed bone and active ossification foci.
Discussion
Autogenous graft harvesting site morbidity is a significant issue, and the incidence may be underestimated [15]. Other substitutes such as hydroxyapatite [30–33] and other ceramics have had variable results when used for spinal fusion or other orthopedic applications. Disease transmission through allograft though remote, still remains a possibility. The effectiveness of bone morphogenetic proteins (BMPs) has been shown in human clinical trials. But high cost, unknown potential long-term biological effects, and issues with carriers and dosing may alter widespread clinical use, at least in the short term [16, 27].
Autogenous bone marrow aspirate has been successfully used in the treatment of congenital and post-traumatic pseudarthrosis [7, 8, 11, 25, 28]. In 1997, Muschler et al. [24] studied the prevalence and concentration of osteoblastic progenitors in marrow aspirates from the anterior iliac crest of 32 patients without systemic disease. They determined the number of alkaline phosphatase-positive colony-forming units that grew after placing the bone-marrow derived cells into tissue-culture medium. Three important conclusions have been drawn from this study: (1) the authors estimated that 80% of the cells found in the first 2 ml of aspirated bone marrow have a prevalence of alkaline phosphatase-positive colony-forming of one for 35,000 nucleated cells; (2) the bone marrow derived cells concentration decreases with the volume of aspirate due to dilution; (3) the cellularity of bone marrow and the prevalence of osteoblastic progenitor cells significantly differ between humans. The prevalence of osteogenic precursors cells, can be increased by centrifugation [7] or using a three-dimensional structure to which these cells can attach [25]. MCM is such a three-dimensional matrix.
As described previously, implant removal was performed usually as soon as there was radiological evidence of vertebral body fracture healing and the patient could return to all-day activity, but not earlier than 12 months after surgery. Patient 2, who suffered from a polytrauma, underwent repeat surgery for his open pilon fractures and was bound to a wheelchair for a long period. This is the reason why implant removal has been done later than in other patients, almost 2 years after index surgery (20 months).
Conclusions
We conclude that this study shows some evidence that this MCM graft may be a valid bone substitute for posterior or posterolateral spinal fusion under favorable conditions: presence of bone marrow cells, presence of healthy bleeding bone and mechanical stability (provided by the AO fixateur interne in these cases). A larger patient series and/or randomized controlled studies are warranted to confirm these initial results.

fusion
fracture
bone graft substitute
thoracolumbar spine