Distinguishing benign notochordal cell tumors from vertebral chordoma

Objective The objective was to characterize imaging findings of benign notochordal cell tumors (BNCTs).
Introduction
The giant notochordal rest and notochordal hamartoma are terms that have been used to describe a benign notochordal cell tumor (BNCT) that may be mistaken for a chordoma [1, 2]. This recently discovered intraosseous benign lesion of notochordal cell origin may be a potential precursor of chordoma [3–6], but based on the few cases with long-term follow-up (those previously published and our present series) none has clinically grown to become a chordoma. The anatomical predilection is identical to that of chordoma. The most common locations are the sacro-coccygeal region and the base of the skull, followed by cervical and lumbar vertebrae. Most tumors are usually small and asymptomatic and are often found at autopsy. The occasional lesion may become large and can be detected on advanced imaging. Our working group collected 7 cases of BNCT that were detected on imaging. We report on their management, outcomes, and histology. Five patients underwent surgical management based on a misdiagnosis of chordoma and failure to recognize the entity of BNCT. This paper focuses on the clinical, radiological, and histological features of these cases, which get overtreated when misdiagnosed.
Materials and methods
The data came from 7 patients with BNCTs that were found clinically. Clinical records included patient sex, age, presenting symptoms, and anatomic site involved. All formalin-fixed specimens were decalcified with formic acid solution and then routinely processed for hematoxylin-eosin and periodic acid Schiff (PAS) staining. For immunohistochemical study, paraffin-embedded specimens were cut into 3- to 5-μm-thick sections and then mounted on glass slides coated with 3-aminopropyltriethoxysilane. The sections were stained by the streptavidin-biotin-peroxidase method (Nichirei, Tokyo, Japan). The antibodies used are listed in Table 1. Microwave antigen retrieval was carried out prior to immunostaining with vimentin, AE1/AE3, CAM 5.2, and cytokeratin (CK) 18. To determine the specificity of immunostaining, we included known positive and negative tissues as controls.
Table 1Antibodies used in the studyAntibodiesSourceClonalityDilutionEpithelial membrane antigenDAKO Cytomation, Glostrup, DenmarkM1:100Cytokeratin (AE1/AE3)DAKO Cytomation, Glostrup, DenmarkM1:100Cytokeratin (CAM5.2)Becton Dickinson, San Jose, CA, USAM1:1Cytokeratin 18DAKO Cytomation, Glostrup, DenmarkM1:20VimentinDAKO Cytomation, Glostrup, DenmarkM1:100S-100 proteinNichirei, Tokyo, JapanP1:200M, monoclonal; P, polyclonal
Results
Table 2 summarizes ages, symptoms, treatment, and outcomes of the 7 patients with BNCTs that were found clinically (Figs. 1, 2). Two lesions were documented as possible vertebral osteonecrosis without collapse; however, recent histological re-evaluation indicated BNCTs [7, 8]. The other cases were originally diagnosed as chordoma histologically. The patient group consisted of 3 men and 4 women ranging in age from 22 to 55 years (mean, 44 years). One patient was found to have two lesions at different sites, evident in the fifth lumbar vertebra and the second sacral vertebra (Fig. 3). The latter was not examined microscopically. A small lesion was found incidentally in another patient in the far distal coccyx and was resected. Of the 9 lesions, 4 were in the cervical vertebra; 2 each in the lumbar vertebra and sacrum; and 1 in the coccyx. The most common symptom was pain in 5 patients; however, 1 patient complained of pain radiating to the fingers and numbness that possibly was caused by known ossification of the posterior longitudinal ligament (OPLL) at the sixth to seventh cervical vertebrae. Another patient was found to have cervical OPLL on radiographs. The pain was usually indolent except for that caused by OPLL. Lesions were found incidentally in 2 patients on MR images when they were examined for unrelated symptoms due to traffic accidents. A needle or open biopsy was done for 5 lesions. Four lesions were diagnosed as chordoma and 1 as osteonecrosis. The remaining 2 lesions were clinically diagnosed as osteonecrosis and an intraosseous benign condition respectively. The patients whose lesions were diagnosed as osteonecrosis and chordoma respectively underwent curettage. Three patients underwent vertebrectomy after the diagnosis of chordoma. One patient underwent wide resection following a preceding curettage. One patient who did not undergo a surgical procedure was followed because the diagnosis of osteonecrosis was made at biopsy. Another patient did not undergo any surgical management in spite of the histological diagnosis of chordoma at biopsy and was followed because the clinician doubted the histological diagnosis of chordoma based on unusual radiological findings for chordoma. The prognosis of all patients but 1 was excellent. Four patients who underwent surgical management were well without recurrent or metastatic disease for follow-up periods between 17 and 84 months (mean 54.5 months). Two patients who did not undergo surgery were alive without progressive disease, as judged by imaging, for 13 months and 44 months after biopsy respectively (Fig. 4). One patient who underwent vertebrectomy died of hemostasis after surgery because she would not accept a blood transfusion on religious grounds.
Table 2Summary of benign notochordal cell tumors found clinicallyPatientAge (years)GenderLocationSymptomsTreatmentOriginal diagnosisFollow-up information130MaleL4Low back painVertebrectomy following biopsy and curettageChordomaAlive with no evidence of disease, 60 months253FemaleC6Numbness and pain of the fingers bilaterally, probably due to OPLL at C6, 7Vertebrectomy following biopsyChordomaAlive with no evidence of disease, 57 months355FemaleC5Neck pain, shoulder stiffnessBiopsyChordomaAlive with no progressive disease, 13 months450MaleC3Car accident injury (incidentally found on MRI)CurettageOsteonecrosisAlive with no evidence of disease, 84 months552MaleC5Mild upper back pain OPLL, focalNeedle biopsyOsteonecrosisAlive with no progressive disease, 44 months622FemaleS4, Cox (microscopic)Buttock painWide resection following curettageChordomaAlive with no evidence of disease, 17 months743FemaleL5, S2Car accident injury (incidentally found on MRI)Vertebrectomy following biopsy at L5ChordomaDied of postoperative complicationsL, lumbar vertebra; C, cervical vertebra; S, sacrum; Cox, coccyx; OPLL, ossification of posterior longitudinal ligamentFig. 1A 30-year-old man who complained of low back pain (patient 1). a Lateral radiograph of the lumbar spine reveals vague sclerosis in the fourth lumbar vertebra. b Axial computed tomography (CT) scan of the fourth lumbar vertebra demonstrates significant sclerosis in the center of the body, partly extending to the cortex. c Sagittal T1-weighted spin echo magnetic resonance (MR) image reveals a large intraosseous lesion with low signal intensity. The normal bone marrow signal is preserved in the anterior and posterior portions of the body. d Sagittal T2-weighted MR image shows slightly bright signal intensity intermingled with intermediate signal. e Sagittal gadolinium-DTPA-enhanced MR image does not demonstrate any enhancement. No soft tissue mass is recognizedFig. 2A 53-year-old woman who was examined for ossification of the posterior longitudinal ligament at the sixth to seventh cervical vertebrae (patient 2). A lesion was found incidentally. a Lateral radiograph reveals faint or vague osteosclerosis in the 6th cervical vertebral body. The physiological lordosis of the cervical spine is decreased. b Axial CT scan reveals diffuse sclerosis within the vertebral body. c Sagittal T1-weighted spin echo MR image reveals low signal intensity in almost the entire marrow space of the sixth cervical vertebra. d Sagittal T2-weighted MR image reveals intensely high signal replacing the entire marrow space. No soft tissue mass is evidentFig. 3A 43-year-old woman who was found to have two separate abnormalities in the lower spine during an imaging study for a traffic accident (patient 7). a Lateral radiograph of the lumbar spine and sacrum reveals intense sclerosis of the entire fifth lumbar vertebral body and mild sclerosis in the cephalad portion of the sacrum. b Sagittal T1-weighted MR image reveals homogeneous low signal intensity in both the fifth lumbar and second sacral vertebraeFig. 4A 52-year-old man who complained of mild upper back pain (patient 5). a Sagittal T2-weighted MR image reveals an intraosseous lesion with high signal intensity in the fifth cervical vertebra at initial presentation. b Sagittal T2-weighted MR image demonstrates no progressive disease 14 months after needle biopsy. No extraosseous tumor extension or enlargement is recognized
Imaging findings
Clinical images reviewed included conventional radiographs (n = 9), bone scintigrams (n = 2), CT scan (n = 7), and MR imaging (n = 8). Conventional radiographs revealed ill-defined, vague sclerosis within the vertebral body in 5 lesions (Figs. 1, 2) and diffuse prominent sclerosis presenting as ivory vertebra in 1 (Fig. 3). The remaining 3 lesions were not identified on radiographs. In all 4 lesions involving the cervical spine, physiological lordosis was decreased. Bone scintigrams did not reveal any abnormal uptake.
All lesions were sclerotic on CT. One was mildly sclerotic in the center of the vertebral body. The others were moderately to markedly sclerotic (Figs. 1, 2). Among them, 5 lesions that were located in the central part of the vertebral body extended partly to the cortex. Another lesion had diffuse sclerosis replacing the entire bone marrow space. No cortical disruption or bone destruction was evident in any of the lesions.
T1-weighted MR images revealed an intraosseous lesion with homogeneous low signal intensity (Figs. 1–3). T2-weighted MR images revealed homogeneous intermediate to bright signal intensity. Gadolinium-DTPA-enhanced T1-weighted MR images were available for 3 lesions and did not show any significantly increased intensity (Fig. 1). Three lesions occupied the entire marrow space and the other 5 lesions were surrounded by residual bone marrow tissue. No extraosseous disease was demonstrated.
Histological findings
Eight lesions were histologically examined. All lesions were located within the vertebral bodies. The lesions were unencapsulated and composed of solid sheets of adipocyte-like vacuolated chordoid cells mixed with eosinophilic, less vacuolated cells of various degrees (Fig. 5a). The adipocyte-like cells had clear cytoplasm and eccentrically or centrally located round or polygonal nuclei (Fig. 5b). Some tumor cells were multi-vacuolated and their nuclei were mildly polymorphic or hyperchromatic (Fig. 5c). No mitotic figures were recognized in any of the lesions. No myxoid background was evident. In some cases, cystic spaces were found in the lesions that contained eosinophilic colloid-like material. The lesions lacked any apparent vascular network. Islands of hematopoietic bone marrow were seen within the lesions. The entrapped bone trabeculae were sclerotic because of appositional or reactive new bone formation (Fig. 5a,b). PAS staining demonstrated intracytoplasmic glycogen granules. In all 7 lesions examined immunohistochemically, the tumor cells stained intensely positive for vimentin, S-100 protein, EMA, CAM5.2, AE1/AE3, and CK18 (Fig. 5d,e). The appearance in each of the cases was consistent with benign notochordal cell tumor [4, 5]. There were no features to support the diagnosis of chordoma. Patient 7 was presented at the Members Meeting of the International Skeletal Society in 1999.
Fig. 5Photomicrographs of benign notochordal cell tumors. a Low power magnification (patient 1) reveals solid sheets of adipocyte-like vacuolated cells. The affected bone trabeculae are sclerotic. Some islands of non-neoplastic hematopoietic bone marrow are seen in the lesion (hematoxylin-eosin [HE] stain). b Higher power magnification (patient 1) reveals a solid sheet of vacuolated tumor cells of various sizes. The nuclei are oval and appear bland (HE stain). c Some tumor cells (patient 1) are multivacuolated and centrally located nuclei are mildly polymorphic. They are reminiscent of lipoblasts (HE stain). d Biopsy specimen (patient 5) reveals sheets of vacuolated cells with pyknotic nuclei between bone trabeculae. The tumor cells may be mistaken for degenerative fatty marrow (HE stain). e The tumor cells (patient 5) stain immunohistochemically positive for cytokeratin (AE1/AE3) indicating notochordal cell origin (SAB immunohistochemical stain)
Discussion
Six lesions consistent with BNCT have been documented previously by others [1, 2, 9–11]. Darby and coauthors [1] first described a case in 1999, which was surgically excised. A 39-year-old man who complained of a 7-week history of persistent low back pain was found to have an intraosseous lesion replacing almost the entire fifth lumbar vertebra. Radiographs and scintigraphy appeared normal. The lesion was well visualized by MRI, but showed only mild sclerosis on a CT scan. Histological findings were identical to those of our cases presented here. They were not typical for chordoma; however, the authors suspected that the lesion might be an intraosseous chordoma rather than a giant notochordal rest. Mirra and Brien [2] reported 2 cases under the name of giant notochordal hamartoma of intraosseous origin. A 42-year-old woman complained of low back pain after falling down the stairs and was found to have an intraosseous lesion involving almost the entire sixth thoracic vertebral body on MRI. The other case was a 41-year-old man who had suffered from intermittent stiffness and mild pain in the neck. MR images demonstrated an intraosseous lesion involving about 98% of the fifth cervical vertebral body. Neither of the lesions was visible on radiographs. CT scans revealed osteosclerosis in the center of the affected vertebral bodies. The lesions showed low signal intensity on T1-weighted MR images and intermediate to high signal intensity on T2-weighted images and no extraosseous disease. No abnormal uptake was detected on bone scintigrams in either case. The patients did not undergo a surgical procedure after the biopsies. The follow-up examinations after 10 years and 38 months respectively demonstrated no progressive disease and indicated their benign nature. Kyriakos et al. [9] reported a case that involved the fourth lumbar vertebra. An 11-year-old boy with Tourette’s syndrome had a 2.5-year history of repeated low back pain after a playground injury. Radiographs, CT scan, and single photon emission computed tomographic bone scan (SPECT) were normal. MRI revealed an intraosseous lesion with low T1- and high T2-weighted signal intensities within the fourth lumbar vertebral body. Cell block material obtained by a fine needle aspiration biopsy led to the diagnosis of chordoma, and the patient underwent L4 vertebrectomy. On subsequent evaluation, they concluded that the lesion was likely a giant notochordal rest rather than chordoma. The most recently documented case reported by Chauvel et al. [10] was an intraosseous lesion in the fifth lumbar vertebral body. The patient was a 45-year-old woman who had complained of low back pain for 2 years. Radiographs and bone scan were normal; however, CT scan revealed fine sclerosis in the L5 vertebra. MRI demonstrated an intraosseous lesion with homogenous low signal on T1-weighted image and high on T2-weighted image. The patient underwent L5 vertebrectomy because the lesion was histologically diagnosed as a chordoma on biopsy. The histological features from the L5 vertebra were similar to those of previously documented cases reported by Darby et al. [1], Mirra and Brien [2], and Kyriakos et al. [9]. A further case was illustrated in the textbook by Dorfman and Czerniak [11]. The lesion entirely occupied the third lumbar vertebra.
Recent studies have discovered unique benign notochordal cell lesions within axial bone that may be precursors of chordoma [3–6, 12]. The new term “benign notochordal cell tumor” was proposed for these lesions [4, 5]. Careful autopsy examination can identify the lesions in approximately 20% of adult cadavers [4, 5]. They are usually tiny or small and found most frequently in both ends of the axial skeleton followed by the mobile spine. Their histological features are different from those of classic chordoma or notochordal vestiges in fetal intervertebral disks (Table 3). BNCTs are characterized histologically by intraosseous sheets of adipocyte-like vacuolated chordoid cells intermingled with less vacuolated eosinophilic cells of various degrees. The lesions were not lobulated. Some colloid-like material containing cystic spaces are seen in the lesions; however, the lesions lack any myxoid background. The nuclei are usually bland, but sometimes appear mildly atypical. No mitotic figures are recognized. The affected bone trabeculae are sclerotic because of appositional or reactive new bone formation. These histological features are completely identical to those of the lesions reported by Darby et al. [1], Mirra and Brien [2], Kyriakos et al. [9], Chauvel et al. [10], and Dorfman and Czerniak [11]. These reported cases are considered by us to represent BNCTs that grew and replaced almost the entire vertebrae.
Table 3Imaging and microscopic features of benign notochordal cell tumorsEvaluation method FeaturesRadiograph (n = 9)Vague sclerosis (n = 5)Marked sclerosis (n = 1)Invisible (n = 3)Bone scintigram (n = 2)No abnormal uptake (n = 2)CT scan (n = 7)Osteosclerosis in vertebral body (n = 7)No bone destruction or cortical disruption (n = 7)MRI (n = 8)T1-WI (n = 8): low signal intensityT2-WI (n = 8): intermediate–high signal intensityGd-DTPA T1-WI (n = 3): no enhancementNo invasive soft tissue mass (n = 8)Histology (n = 8)Solid sheets of adipocyte-like vacuolated cells combined with less vacuolated eosinophilic cells of various degreesRound or polygonal pyknotic nuclei occasionally with polymorphismNo lobular configurationNo extracellular myxoid matrixSome cystic spaces containing eosinophilic colloid-like materialNo mitotic figuresPoor vascular networkEntrapped islands of hematopoietic bone marrowSclerotic bone trabeculae affectedPositive immunostaining for vimentin, cytokeratin (AE1/AE3, CK18), EMA, and S-100 proteinCT, computed tomography; MR, magnetic resonance; WI, weighted image; Gd; gadolinium; CK, cytokeratin; EMa, epithelial membrane antigen.
Benign notochordal cell tumors are usually asymptomatic or indolent. Most lesions are overlooked, even at autopsy, because of their anatomical location or size. Only one such lesion had been documented with a name of ecchordosis physaliphora vertebralis in 1982 [13]. Enlarged BNCTs may be detected on CT or MR images, or rarely by conventional radiography. The imaging findings of our cases are somewhat variable, but distinctive in the absence of trabecular destruction on CT or of a soft tissue mass on either CT or MR images (Table 3). Gadolinium enhancement was absent in the 3 patients in whom it was administered. Radiographs may reveal ill-defined, vague sclerosis in the vertebral body and occasionally show diffuse sclerosis presenting as an ivory vertebra (Fig. 3). Often the lesions are invisible on radiographs. CT scans may reveal unequivocal sclerosis, which is much more obvious than on radiographs. No bone destruction or cortical disruption is recognized. T1-weighted MR images reveal relatively well-demarcated intraosseous lesions with homogeneous low signal intensity. T2-weighted MR images show homogeneous high signal intensity. Gadolinium-DTPA enhanced T1-weighted MR images do not show enhancement. Bone scintigraphy did not show any abnormal uptake.
Diagnosis of the cases presented was based upon biopsy or preoperative imaging; the diagnosis was chordoma in 5 cases and osteonecrosis in 2. Hence, 5 patients underwent vertebrectomy, wide resection, or curettage. All patients but 1 are well without recurrent or metastatic disease. Two patients who underwent only biopsy had no progressive disease, as judged by radiography, CT, or MR images, for 13 months and 44 months respectively. As described by Mirra and Brien, the follow-up information was consistent with the benign nature of the tumors.
The terminology of the lesions is controversial. Dorfman and Czerniak [11], Kyriakos et al. [9] and Chauvel and coauthors [10] used the term giant notochordal rest. Mirra and Brien [2] proposed the term giant notochordal hamartoma of intraosseous origin. In addition, the International Skeletal Society has used the term benign chordoma on the brochure of the Pathology Refresher Course since 2003. However, we prefer the term “BNCT” to the others. BNCTs show histological and immunohistochemical features that are different from those of notochordal vestiges in fetal intervertebral disks. BNCTs lack any myxoid background and show positive immunoreactivity for cytokeratin 18 although notochordal vestiges in the fetal intervertebral disks are associated with myxoid matrix and are negative for cytokeratin 18. Notochordal vestiges are often found in the intervertebral disks of neonates and finally disappear by the age of 1 to 3 years. In our personal autopsy experience, we have never seen any notochordal tissue in the vertebral bodies in fetuses and babies who had notochordal vestiges in their intervertebral disks. In contrast, BNCTs are found in 20% of the axial skeleton dissected from adult cadavers. Therefore, BNCTs are considered to develop after birth. The term of hamartoma is not proper for the lesions, as mentioned by Kyriakos et al. [9] and one of the present authors [3]. The term of benign chordoma is oxymoronic because “chordoma” has been used to describe a malignant notochordal cell tumor [14].
In 15 clinically identified BNCTs, including our cases and those reported by others, 6 tumors were found in the lumbar spine, 5 in the cervical spine, 2 in the sacrum, and 1 each in the thoracic spine and coccyx. This anatomical distribution is different from that found at autopsy [2]. We believe the reason for this discrepancy is that the cervical and lumbar regions often are examined with MRI for neck pain and low back pain. Lesions in the sacrum and coccyx may be overlooked because they are less frequently imaged.
The most important disease that should be distinguished from BNCT is chordoma. Microscopically, chordoma is composed of cords or strands of atypical chordoid cells with a myxoid matrix of various degrees [11, 15, 16]. Chordomas are associated with a large soft tissue component. However, differential diagnosis may be difficult because cellular atypia of BNCT and chordoma may overlap. They also share the same immunohistochemical profile [3]. Imaging is critical in separating the benign lesion from the chordoma. Chordomas are osteolytic tumors. In contrast, BNCTs may show osteosclerotic reaction and do not exhibit osteolytic change. Chordomas are not intracompartmental lesions and are associated with a soft tissue mass. In those cases with histological overlap, with our present understanding of these diseases, we would suggest that the absence of a soft tissue mass should favor a BNCT and the presence of a soft tissue mass should indicate a chordoma.
Biopsy specimens of BNCTs may be easily mistaken for fatty marrow because of their morphological similarity to that tissue. Indeed, 2 lesions among our cases were overlooked as normal fat marrow on biopsy sections. Careful observation can distinguish BNCTs from fatty marrow. Furthermore, immunohistochemical study can help to distinguish BNCTs from fatty marrow. BNCTs stain positive for epithelial markers whereas fat cells do not (Fig. 5). Differential diagnosis also includes metastatic carcinoma, particularly originating from clear cell carcinoma of the kidney. Metastatic clear cell carcinoma usually is osteolytic on radiographs and CT scans. Gadolinium-DTPA enhanced T1-weighted MR images have increased signal intensity. Histologically, clear cell carcinoma has an alveolar pattern and vascular fibrous septa. Clear cell carcinoma rarely exhibits positive immunoreaction for S-100 protein [17].
Large BNCTs may be found during routine clinical examination. They may have the potential of malignant transformation to chordoma; however, the transformation, as we know from the collected cases, is a rare happening [6]. We, however, are not aware of any cases of what we term BNCT growing beyond the confines of bone and displaying features of chordoma, and therefore they should be managed as benign lesions and carefully followed by thin section CT and MRI for signs of extraosseous disease. More extensive follow-up by cross-sectional imaging over a long period of time would be required before firmly determining the biological behavior of BNCTs. BNCTs do not require any surgical management until they undergo malignant transformation. BNCTs should be recognized by radiologists, pathologists, and orthopedic surgeons to prevent unnecessary radical surgery and carefully followed by advanced imaging.

benign notochordal cell tumor
chordoma
notochordal rest
notochordal hamartoma
vertebra
mri
spine


Second-Look Operation for Unresectable Pancreatic Ductal Adenocarcinoma at a High-Volume Center

Background The value of re-exploration for pancreatic ductal adenocarcinoma after the initial diagnosis of unresectability is unclear.
Surgical resection is the only curative approach available for patients suffering from pancreatic cancer.1 However, resection rates remain low, not only because advanced disease is present in the majority of patients, but also because nonspecialized centers often have insufficient experience with radical surgery.2–4 Resection rates in tertiary referral centers are increasing due to technical progress, improved perioperative management, increasing emphasis on a more-radical approach, such as resection of tumor-infiltrated portal vein, and improved experience in judging resectability.3,5–8 This holds particularly true for evaluating vascular infiltration, which may preclude resection. In these cases, extensive mobilization and careful dissection are necessary to distinguish accompanying inflammatory adhesions from tumor infiltration of retroperitoneal vessels. However, determination of resectability remains challenging, and thus in selected cases less-experienced surgeons are potentially more prone to mistakenly regard a tumor as unresectable.
Since randomized trials on the value of neoadjuvant therapy have not yet been published, it also remains unclear whether—particularly in younger patients—neoadjuvant treatment (or reoperation after initially palliative chemotherapy) may be beneficial, at least in some patients, and may thus lead to resectability of an initially unresectable tumor.
Reoperation of patients who were deemed unresectable may be a further option to increase resection rates.9–18 The value of re-exploration has only been analyzed by a few groups: 10 studies have addressed this question, with the first study concluding that an “appreciable salvage rate” is feasible on re-evaluation.9 Subsequent studies confirmed these results and demonstrated that a second-look operation can be performed with low morbidity and mortality rates, and that this approach confers a survival benefit for the then resectable patients.10–18 Calculations of statistically significant differences in survival of resected and unresected patients at reoperation are hampered by insufficient power of the individual studies due to small patient groups. However, there is a general trend showing that re-exploration may be an option in carefully selected patients at specialized centers.
In this study, we retrospectively analyzed our single-center experience with a group of patients who had initially been diagnosed as unresectable and who were subsequently referred to us for re-exploration. In addition, a literature review of the available (retrospective) studies was performed to more precisely define the value of re-exploration.
PATIENTS AND METHODS
From our database we identified 33 patients with ductal adenocarcinoma of the pancreas who had initially been diagnosed as unresectable (at other institutions) but were re-explored in the course of their disease. Patient characteristics, initial procedures, subsequent palliative or neoadjuvant therapies, operative details of the re-exploration, morbidity, and mortality (death within 30 days after surgery) were prospectively recorded. Patients were followed up until February 2007. At the time of analysis, two out of 14 patients in the group of unresectable patients and 11 out of 19 patients in the resection group were alive.
During the second-look operation, resectability was defined using previously reported criteria.1 If resection was impossible, a tumor biopsy was taken from those patients in whom the initially performed palliative operation was sufficient. In the case of an obstruction, a gastroenterostomy, hepaticojejunostomy or a double bypass procedure was performed. In resectable patients, a pylorus-preserving pancreaticoduodenectomy (ppPD) was the standard operation. However, if the tumor involved the pyloric region, a classical PD was carried out. To achieve tumor-free resection margins, a total pancreatectomy had to be performed in two cases. A distal pancreatectomy was performed for one tumor of the tail of the pancreas.
Pancreatic tumors other than ductal pancreatic adenocarcinoma were excluded. The specimens from resectable (at reoperation) patients were classified according to the tumor, node, metastasis (TNM) classification.19
For the preoperative imaging of operable patients, computed tomography of the abdomen or magnetic resonance imaging/magnetic resonance cholangiopancreaticography (MRI/MRCP) was the standard.
Statistical Analysis
The Kaplan–Meier method was used for presentation of the survival curve, and differences in survival were evaluated with the log-rank test. A P value of < 0.05 was regarded as statistically significant.
Literature Review
A literature search was performed independently by CWM, JK, and JB as described previously.20 The last search was carried out on 30 January 2007. Available manuscripts were cross-searched to identify studies that had not initially been found. The search strategy revealed 10 retrospective studies on re-exploration after initial diagnosis of unresectability. No stratification was performed because only retrospective analyses have been published. Data collection was conducted as previously described.20
RESULTS
In this study, 33 patients (median age 60 years) from our prospectively maintained database who had been re-explored after having initially been defined as unresectable (operation between 2001 and 2005) were included. At the time of the initial operation, an exploratory laparotomy was performed in 51% of the patients, while in the other 49% a bypass operation or a biopsy was carried out (Table 1). The main reason for the definition of unresectability at the initial operation was vascular infiltration (six arterial infiltrations, nine venous infiltrations). In some patients, metastatic disease was present (Table 2). After the first operation, most patients received palliative/neoadjuvant therapy. The types of treatment varied considerably: 13 patients received chemoradiation, 12 patients were treated with chemotherapy, and eight patients received no therapy. There were no standardized treatment protocols for palliative/neoadjuvant therapy, but patients were treated according to local guidelines at the institution where the initial diagnosis had been made.
Table 1.Patient characteristicsUnresectable patients (n = 15)Resectable patients (n = 18)Age60 (41–68)61 (33–71)Female34Male1214Initial operation  Explorative laparotomy48  Gastroenterostomy50  Double bypass35  Lymph node biopsy10  Resection of abdominal wall tumor10  Laparoscopy02  Hepaticojejunostomy13Interval between surgeries (median)88 days101 daysPreoperative tumor markers  CA19-9 (median)608 U/l117 U/l  CEA (median)39143 U/l136 U/lDiabetes mellitus36Neoadjuvant therapy1213Table 2.Criteria for initial inoperabilityUnresectable patients (n = 15)Resectable patients (n = 18)Peritoneal metastasis40Vascular infiltration69Liver metastasis31Duodenal infiltration11Lymph node infiltration14Other/unknown03
Upon referral to our department, all patients presented in adequate general condition (ASA score ≤ 3; American Society of Anesthesiologists). At the second operation, 18 patients were found to have a resectable tumor, while in 15 patients the primary tumor could not be resected. The median time from initial surgery to re-exploration was 88 days in the unresectable group and 101 days in the group of patients in whom a resection could be performed. Except for one patient with a large tumor infiltrating the retroperitoneal vessels, metastatic disease (not detectable in the abdominal CT scan) was present in all the unresectable patients, precluding a surgical resection (Table 4).
In the group of patients who were found to be resectable, 15 pancreaticoduodenectomies, two total pancreatectomies and one distal pancreatectomy were performed (Table 3). Portal vein/superior mesenteric vein (SMV) resections had to be carried out in four patients. There was one patient with a liver metastasis at the initial operation (Table 2) who was resected at the second-look surgery. The patient had no liver metastases at the second look and was therefore considered resectable. This patient is alive at 1241 days after the initial surgery. Four out of five patients without neoadjuvant treatment who were resected (Table 1) received adjuvant chemotherapy. One patient refused adjuvant treatment.
Table 3.Surgical procedures at second operation, morbidity and mortalityUnresectable patients (n = 15)Resectable patients (n = 18)Exploration with biopsy7–Double bypass3–Gastroenterostomy3–Hepaticojejunostomy2–Pancreaticoduodenectomy–5Pylorus-preserving pancreaticoduodenectomy–10Total pancreatectomy–2Left resection–1Duration of operation (min; median)105440Blood loss (ml; median)100500Morbidity23Delayed gastric emptying10Bilioma10Wound dehiscence01Cholangitis01Lymph fistula01Mortality01Length of stay (days; median)1112Table 4.Criteria for unresectability at reoperationUnresectable patients (n = 15)Peritoneal metastasis6Liver metastasis5Omental metastasis1Peritoneal and liver metastasis2Retroperitoneal infiltration1Table 5.Review of studies on re-exploration for pancreatic cancerAuthorYearInstitutionPatientsResection rateMoosa1979University of Chicago2417/24 (71%)Jones1985University of Toronto50N/AHashimi1989Bradford Royal Infirmary2611/26 (42%)McGuire1991Johns Hopkins Medical Institutions5533/55 (60%)Tyler1994M.D. Anderson Cancer Center1914/19 (74%)Robinson1996M.D. Anderson Cancer Center2929/29 (100%)Johnstone1996Naval Medical Center, San Diego2916/29 (55%)Sohn1999Johns Hopkins Medical Institutions7852/78 (67%)Chao2000Fox Chase Cancer Center4022/40 (55%)Shukla2005Tata Memorial Hospital, India1515/15 (100%)This series2007University of Heidelberg3318/33 (55%)
In the group of unresectable patients, bypass procedures were carried out in eight patients, while seven patients underwent exploration with tumor biopsy (Table 3). The postoperative course of the patients was mostly uneventful, with two morbidities in the inoperable group and five in the resected patients (Table 3). One patient who was resected died in the immediate postoperative period (mortality 1/18). The duration of the operation, amount of blood loss, and length of stay were increased in the resection group (Table 3).
Pathology reporting revealed that a T3 tumor was present in most of the cases. Only one patient was found with a T2 tumor. Nine pancreatic specimens (50%) were node positive, with an average of 21.5 harvested lymph nodes. A grade 1 tumor was found in only two patients, whereas a grade 2 tumor was present in nine patients and a grade 3 pancreatic cancer was present in seven patients.
The Kaplan–Meier survival curves revealed a median survival of 439 days for unresectable patients versus 934 days for resected patients (after the second operation). A log-rank test demonstrated that this difference was statistically significant (P = 0.013; data not shown). To better judge the influence of neoadjuvant/palliative treatment on survival, a Kaplan–Meier curve was calculated for the survival after the initial operation. This analysis showed a significantly different median survival of 547 versus 1078 days for unresectable versus resectable patients (P = 0.018; Fig. 1).
FIG. 1.Kaplan–Meier survival curve of re-explored patients after initial operation. Comparison of the survival curves of re-explored, resected patients (n = 17; one excluded due to postoperative mortality; red) and re-explored, unresectable patients (n = 15; black). A log-rank test demonstrated significantly increased survival in the resected patients (P = 0.018).
To judge whether resection at reoperation influences the outcome, we compared re-resected (18) and non-re-resected (15) patients with a cohort of 554 patients who were resected at the initial operation at our center and with a cohort of 250 patients who could not be resected. The Kaplan–Meier survival curve followed by a log-rank test revealed no differences in survival (P = 0.2514; Fig. 2) when comparing initially resected patients and patients resected at the second operation. Furthermore, there were also no differences in comparison of the unresectable patients, irrespective of the second surgery (P = 0.45; Fig. 3).
FIG 2.Survival curves of re-explored, resected patients and 572 primarily resected pancreatic ductal adenocarcinoma (PDAC) patients. Comparison of the survival curves of re-explored, resected patients (n = 18; red) and a control cohort of 572 patients (black) who were initially resected revealed no differences regarding survival (P = 0.2514).FIG. 3.Overall survival of re-explored, unresectable patients and 256 unresectable patients. The survival of a control cohort of 256 PDAC patients who were unresectable (n = 265) was compared with re-explored, unresectable patients (n = 15; red), revealing no differences in survival (P = 0.45).
Subsequently, we performed a retrospective review of the causes of initial unresectability. This revealed that only 11 of 33 patients had initially been treated at a high-volume center (defined as hospitals with a case load of >16 pancreatic resections per year7). Except for four cases with metastasized disease, infiltration of local structures was the main reason for initial unresectability. A detailed analysis of these causes of unresectability showed that 14 of 33 patients would probably have been resected if they had initially been referred to our center. In these cases, mainly venous (SMV/PV) infiltration was present, which may not be considered a contraindication for a resection.
Review of Retrospective Studies
Ten studies on the value of re-exploration to potentially achieve resection have been published so far.9–18 In the first study, by Moosa,9 17 patients were re-evaluated after having initially been diagnosed as unresectable. Of these, nine patients underwent total pancreatectomy, while in two patients no tumor was detectable. The survival of the resected cohort was 1.5–6 years, which was higher than the mean survival in the group of patients with inoperable disease (7–12 months). Similar results were reported by Hashimi et al., who performed a second-look operation on 30 patients, revealing that nine were resectable, while in two patients no tumor was found despite several biopsies.11 In the study by Jones and co-authors, preliminary surgery (nonresective) had been carried out before referral in 51% of the patients, leading to the correct diagnosis in only 44% of these.10 However, outcomes for this patient cohort are not specifically described. Two publications from the Johns Hopkins Institutions also extensively describe results obtained in re-exploratory surgery of two patient cohorts (recruited at different time periods: from 1979 to 1990 and from 1991 to 1997). In the first publication,12 the resection rate for pancreatic cancer at re-exploration was 58%, with subsequent mean survival periods of 20.5 months (pancreatic adenocarcinoma) and 33 months (nonpancreatic periampullary malignancies), which were comparable to the cohort of initially resectable patients. Sohn et al. subsequently described results obtained from re-exploration of a second cohort of patients that included all periampullary tumors.16 The resection rate at reoperation was 67%. There was a striking survival difference when comparing the unresectable and the resectable groups (at re-exploration), with a median survival of 7 versus 23 months, respectively. Comparison of the patients who were resected at re-exploration with a control group who had initially been resected revealed significantly increased survival rates in the reoperative group (Kaplan–Meier survival curve generated from the time of initial operation: 20 months in the control group versus 33 months in the reoperative group, P = 0.02). In the study by Tyler et al. from 1994, reoperation rates were comparable with the earlier published studies (14 out of 19 patients who underwent re-laparotomy were resected), as were the (low) complication rates (three out 14 resected patients).13 At the time of median follow-up of 26 months, four of ten patients with pancreatic adenocarcinoma were alive, suggesting survival rates comparable to previously published results. Similarly, Johnstone and Sindelar showed a resection rate of 55% at re-exploration and significantly improved disease-specific survival rates in the resected group (P < 0.01) 14. Robinson and co-workers demonstrated an exceptionally high resection rate at reoperation of 100%.15 In a study by Chao and co-workers, a resection rate at the second look operation of 55% was shown.18 Furthermore, they could demonstrate that resection at the second operation led to “a median survival comparable with that of patients who did not undergo previous exploration”. In the most recently published study, by Shukla et al., all patients who were reoperated could be resected.17 Confirming the previously published studies, low morbidity and mortality rates were demonstrated.
DISCUSSION
This single-center experience shows that re-exploration of patients initially deemed unresectable may be an option for the treatment of pancreatic cancer. In concordance with previously published studies (which were all retrospectively conducted), re-exploration can yield resectability rates greater than 50% and can be performed with low morbidity and mortality. However, whether reoperation confers a survival benefit is still a matter of discussion.
Although diagnosis of resectability has been facilitated by the use of multidetector, high-resolution computed-tomography exploration often remains necessary.21,22 Particularly when neoadjuvant therapy has been applied, fibrosis and an inflammatory reaction frequently hamper exact assessment of tumor size and (potential) metastatic disease. A number of studies have demonstrated that both the complication rate and mortality after pancreatic surgery are significantly higher in hospitals that perform few operations.3,7,8 We found that, upon re-exploration, more than half of the tumors were resectable, and that 42% of the patients initially deemed unresectable could have been resected at high-volume centers. These results support the concept that patients with pancreatic cancer should be treated with an interdisciplinary approach at large tertiary referral centers.
Furthermore, resection in a second-look operation was shown to potentially confer a survival benefit, although it remains unclear whether this is due to resection or to favorable tumor biology in the patients in whom a regression of the initially metastasized or infiltrating tumor was achieved. Altogether, there was significantly increased survival in the reoperated group of patients compared with a control cohort of patients who were not reoperated (resected and unresectable). These results suggest that there might be a generally favorable tumor biology in this group of reoperated patients. However, the differences in survival conferred by resection at reoperation confirm the notion that in a subgroup of patients in good condition, re-laparotomy (and potentially resection) should be attempted. Furthermore, reoperated but not resected patients do not fare worse than those who were not resected initially and who were not explored again after chemotherapy or chemoradiation. Therefore, we again conclude that in a selected cohort of patients with a tendency towards remission, re-laparotomy may be a valid option. This holds particularly true when patients have initially been considered unresectable at smaller hospitals with less experience in pancreatic surgery and especially with less experience in judging whether a tumor is resectable. This is supported by many studies on reoperative pancreaticoduodenectomy for pancreatic cancer.12,13,15–17 As stated by Robinson and co-authors, “detailed preoperative imaging and a clearly defined operative plan would have allowed successful resection at the initial operation” in the majority of patients. However, accompanying inflammatory changes allow judgment of resectability only during the surgical procedure itself in many cases. Therefore, not only preoperative assessment but also the extension of resectability criteria (as present in high-volume centers) is a key factor for improving the outcome in patients with pancreatic cancer. We strongly suggest that all patients suffering from pancreatic cancer should be referred to high-volume centers. Furthermore, patients in generally good condition but locally unresectable disease should receive neoadjuvant chemoradiation, preferably within randomized controlled clinical trials.23 However, we do not consider infiltration of the SMV/portal vein (in contrast to superior mesenteric artery (SMA)/celiac trunk involvement) a contraindication for resection. Following the last cycle of chemoradiation, high-resolution CT will allow judging resectability in many cases and in the absence of metastatic disease, surgical exploration remains the only option to determine resectability.
In conclusion, reoperation for pancreatic cancer after initial classification of unresectability revealed resectability in half of patients. Since the majority had received chemotherapy/chemoradiation after the initial operation, the concept of selecting patients by neoadjuvant therapy may be supported. Furthermore, a subgroup analysis revealed that a large number of patients who were initially deemed unresectable at smaller hospitals would have been resectable at a large tertiary referral center, which again promotes the concept of patient centralization in pancreatic surgery. A survival benefit for the resected patients underlines the efficacy of a surgical resection even in a situation in which the initial findings preclude a potentially curative approach.

unresectability
pancreatic cancer
reoperation
second look


An Ex Vivo Study of the Biological Properties of Porcine Aortic Valves in Response to Circumferential Cyclic Stretch

Normal physiological mechanical forces cause constant tissue renewal in aortic valve leaflets (AVL) while altered mechanical forces incite changes in their structural and biological properties. The current study aims at characterizing the remodeling properties of AVL subjected to cyclic circumferential stretch in a sterile ex vivo bioreactor. The leaflets cultured were stretched at a maximum rate of 300%s−1 corresponding to a 15% strain for 48 h. Collagen, sulfated glycosaminoglycan (sGAG), and elastin contents of the stretched, fresh, and statically incubated leaflets were measured. Cusp morphology and cell phenotype were also examined. AVLs exposed to cyclic stretch showed a significant increase in collagen content (p < 0.05) when compared to fresh and statically incubated AVLs. sGAG content was significantly reduced in the stretched AVLs (p < 0.05) when compared to the fresh leaflets and was comparable between stretched and statically incubated AVLs. There was no statistically significant change in elastin content in all the three groups of AVLs (p > 0.05). Native aortic valve morphology was well preserved in stretched leaflets. Immunohistochemistry and immunoblotting studies showed an increased expression of α-smooth muscle actin (α-SMA) in stretched leaflets while α-SMA expression was reduced in statically incubated AVLs when compared to the fresh leaflets. To conclude, circumferential cyclic stretch altered the extracellular matrix remodeling activity of valvular cells, and consequently the extracellular matrix composition of the AVLs. Most interestingly, the contractile and fibrotic phenotypic expression of valve interstitial cells was enhanced. These results show that circumferential cyclic stretch is a possible mediator for AVL remodeling activity.
Introduction
Cardiac valves play a crucial role in controlling blood flow through the body.6 Of particular interest in this study is the aortic valve, which at its most basic level controls blood flow between the left ventricle and the systemic circulation. In the United States, aortic valve disease has emerged as the third most common cardiovascular disease,17 with the number of aortic valve replacement surgeries steadily increasing over the past 20 years.33
The aortic valve is subjected to 30–40 million cycles of opening and closing a year, which translates to approximately 3 billion cardiac cycles in a lifetime.26 During each cardiac cycle, the normal functional aortic valve interacts closely with the surrounding hemodynamic environment and is exposed to a myriad of mechanical forces such as transvalvular pressure, axial, shear and bending stresses, and cyclic flexure.26 Normal hemodynamic forces have been shown to cause constant tissue renewal in aortic valves,19 while altered mechanical forces are believed to induce changes in aortic valve biology possibly leading to valvular disease.16,30 However, the cellular and molecular events involved in these processes are not well characterized. Studies indicate that abnormal hemodynamics, especially hypertension, experienced by the valve leaflets cause tissue inflammation, which can lead to calcification, stenosis and ultimately valve failure1,15,23Knowledge of the adaptive mechanisms of the aortic valves to altered hemodynamics will therefore improve our understanding on mechanotransduction of valvular disease progression and valve failure.
Mechanical forces modulate cell physiology, and have been shown to affect the biosynthetic activity of cells in tissue matrices. Previous studies on aortic valve mechanobiology have shown that porcine aortic valves cultured under normal hemodynamic conditions in a sterile ex vivo flow loop undergo constant renewal to maintain their native phenotypes.13 Porcine aortic valve endothelial cells exposed to 20 dyn cm−2 of steady laminar shear stress showed an organization of focal adhesion complexes and a cell alignment different than in static endothelial cell culture.4 Aortic valve leaflets exposed to hypertensive cyclic pressure showed increased collagen and sulfated glycosaminoglycan (sGAG) synthesis which varied in a magnitude and frequency-dependent manner.31 Studies exposing native porcine aortic valve leaflets to the isolated effects of flow, laminar steady shear stress, and constant static and cyclic pressure resulted in a reduced level of expression of α-smooth muscle actin (α-SMA).29,31,32 This last finding might indicate the involvement of other forces in maintaining and regulating the contractile phenotype of aortic valve interstitial cells. To date the effect of cyclic stretch on aortic valves has not been studied.
The mechanical mode of cyclic stretching is important for normal aortic valve functionality as it allows, among other things, the valve leaflets to extend and form a coaptive seal with each other. However, it was seen that the valve leaflet tissue becomes less extensible and loses its ability to stretch rapidly with increasing age.15 This is mainly because collagen fibrillogenesis proceeds to maturity by increasing the diameter of some of the constituent collagen fibrils that require greater force to produce the same leaflet extension.8 Although the valve leaflet undergoes biaxial stretching in vivo, studies that investigate the effects of separate uniaxial (circumferential and radial) as well as biaxial cyclic stretch on the aortic leaflets would provide useful information on valve functionality or failure.
We hypothesize that the regulation of extracellular matrix remodeling as well as the expression of the contractile phenotype of aortic valve cells depend on the loading forces due to cyclic stretch. Here, we examined this hypothesis with a focus on the effect of physiological uniaxial circumferential cyclic stretch on the biosynthetic response of aortic valve leaflets using porcine aortic valve leaflets and an ex vivo tensile stretch bioreactor. Results from the current study will improve our understanding of the biological responses of native valves to circumferential stretching and will provide a good basis of comparison for conducting studies on radial cyclic stretching.
Material and Methods
Tissue Harvest
Fresh porcine aortic valves were obtained from a local abattoir (Holifield Farms, Covington, GA) following on-site dissection of the hearts within 30 min of slaughter. The valves were then transported to the laboratory in sterile, ice-cold Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, St. Louis, MO).
Upon arrival to the laboratory, the three leaflets were excised from each valve and a rectangular section of tissue was isolated from the center of each leaflet. This tissue section was further divided into three rectangular sections having an aspect ratio of 12 × 5 mm and oriented in the circumferential direction from the basal region. These three sections from each leaflet were randomly selected as fresh control, static control, and stretched leaflets, respectively (Fig. 1). Leaflet sections from the fresh control group were analyzed within 4 h following valve dissection. Leaflet sections from the static control group were statically incubated in Dulbecco’s Modified Eagle Medium (DMEM; Sigma) at 37°C for the duration of the experiment (48 h). Leaflet sections from the stretched group had loops sutured to the 5 mm ends and were suspended in DMEM in a tensile stretch bioreactor.10 These samples were then cyclically stretched at a frequency of 1.167 Hz (equivalent to 70 beats per minute) to 15% strain at a maximum strain rate of 300%s−1 at 37°C. The temporal strain waveform imposed (Fig. 2a) in the circumferential direction was derived from the variations of the leaflet length in the circumferential direction over one cardiac cycle.25,26 The extension of the leaflet corresponded to diastole (2/3 of the cardiac cycle), while the unstretched state of the leaflet corresponded to systole (1/3 of the cardiac cycle). The gradients of the extension and relaxation approximated those experienced in vivo.25 The above waveform resulted in a tissue extension of 1.8 mm at a maximum rate of extension of 36 mm s−1. Each experiment was run for 48 h, with 12–15 leaflet sections for each experimental run.
Figure 1.Preparation of aortic valve leaflets for the experiment. Three sections were cut out in the circumferential direction from the base, belly and tip regions of the aortic valve leaflet. These sections were randomly chosen as stretched, fresh and static leaflet groups. Fresh sections were processed immediately. Static control sections were incubated for 48 h in DMEM. Stretched sections were stretched for 48 h.Figure 2.(a) Loading curve used in this study. A near physiologic loading curve 25 was input to the actuator to stretch the leaflets. (b) Ex vivo tensile stretch bioreactor used in this study. A magnified image of the tissue chamber is shown on the right showing eight tissue wells, with aortic valve leaflets in four of the wells.
Stretch Bioreactor System and Validation
The stretch bioreactor used in this study (Fig. 2b) was developed by Engelmayr et al. 10The device consists of two tissue chambers coupled via a cross-arm to a centrally positioned linear actuator (Ultra Motion LLC, Mattituck, NY). Each chamber consists of eight culture wells within which two “stationary posts” are positioned. The linear motion of the actuator is translated into uniaxial motion of two “moving posts” via the cross-arm which bifurcates to two moving arms. These moving arms then enter each individual bioreactor chamber and terminate at each culture well with the two “moving posts” mentioned above. The interior of the chamber is protected from external contamination by a neoprene-lined lid. The bioreactor was constructed from polysulfone, which is thermally and chemically stable, while the lids were constructed using acrylic. The device was assembled using 18-8 stainless steel screws (McMaster Carr, Cleveland, OH) and fabricated by Apollo Precision Inc. (Plymouth, MN). The motor input waveform (Fig. 2a) was downloaded to the actuator controller card using the Si-Programmer software (Ultra Motion LLC).
After assembly, the bioreactor was validated for uniformity of strains on the leaflet sections in all the culture wells. This was achieved by placing dye markers (Shandon tissue marking dye, Thermo Electron Corp, Pittsburgh, PA) on aortic valve leaflets and capturing the distance between the markers under uniaxial cyclic stretch using a high-speed camera capture technique developed in our laboratory.12The bioreactor was validated at input strains of 15 and 25%.
In order to determine if the stretch bioreactor induced a shear stress on the leaflet surface due to the relative motion of the leaflet with respect to the surrounding media, a computational fluid dynamics (CFD) model was designed, validated and used to predict the leaflet wall shear stress under the imposed loading curve in the stretch bioreactor.
For the experiment, the bioreactor was sterilized using hydrogen peroxide in a gas-plasma sterilizer (Advanced Sterilization Products, Irvine, CA), and assembled aseptically in a sterile laminar flow hood (Fisher Hamilton, Two Rivers, WI). The culture wells were filled with 8 ml of serum-free DMEM supplemented with 50 mg l−1 ascorbic acid, 3.7 g l−1 sodium bicarbonate, 1% (v/v) nonessential amino acid solution, and 1% (v/v) antimycotic-antibiotic solution (all reagents from Sigma). Each tissue section was suspended in the medium by the two sutured loops – one end was looped over the stationary posts while the other end was looped over the moving post. The bioreactor was then placed in an incubator (Fisher Scientific, Hampton, NH) maintained at 37°C, 5% CO2 atmosphere at normal atmospheric pressure and the leaflets were stretched for 48 h.
Tissue Processing
At the end of the experiment, each leaflet section was further cut into three equal rectangular sections. These resulting samples were dehydrated for 48 h at room temperature in a vacuum oven (Yamato Scientific America, San Francisco, CA) prior to digestion in pepsin (Sigma), protease (Sigma) and proteinase K (Sigma), for analyzing collagen, sulfated glycosaminoglycan (sGAG), and elastin contents, respectively. All digestions were carried out in a water bath maintained at 37°C. The total amount of collagen, sGAG and elastin was determined using the SircolTM Collagen assay kit (Biocolor, UK), BlyscanTM sGAG assay kit (Biocolor), and FastinTM Elastin assay kit (Biocolor), respectively. These kits used quantitative dye-binding methods to determine the total quantity of the respective ECM component in the sample.
Valve leaflet samples from additional experiments were isolated for analysis of tissue morphology and cell phenotype. For histological and immunohistochemical analyses, leaflet sections were fixed in 10% neutral buffered formalin (Fisher Scientific, Suwanee, GA) for 24 h, saturated in 70% ethanol, processed in a tissue processor (Shandon Pathcenter enclosed Tissue Processor) in descending grades of ethanol, embedded in paraffin and cut into 5 μm sections.
Morphological Characterization
Routine Hematoxylin and Eosin (H & E) stain was used to examine gross tissue structure and indicate any changes in the structural compositions of the leaflets. H & E staining was done on an autostainer (Leica, Vashaw Scientific, Norcross, GA). Picrosirius red staining was performed to examine collagen fiber structure and morphology. The protocol was as follows. Deparaffinized slides were incubated in Weigert’s hematoxylin (Sigma) followed by picrosirius red solution for one hour. The slides were then washed in acidified water, three changes of 100% ethanol, and xylene before being coverslipped. These slides were viewed at 100× using a Nikon imaging microscope (Nikon Inc., Melville, NY). A Q-imaging camera, Retiga 1300C (I-Cube, Glen Burnie, MD) and its bundled software Q-Capture Pro was used to acquire images of the tissue sections. H & E images were recorded using normal white light while picrosirius red images were recorded with circularly polarized light.
Characterization of Cell Phenotype
α-smooth muscle cell actin (α-SMA) immunohistochemistry (IHC) was used to detect myofibroblasts and smooth muscle cells. The staining provided information on changes in cell phenotype between the three experimental treatments. The protocol was as follows. Deparaffinized slides were blocked using 1% gelatin/phosphate buffered saline (PBS; Sigma) for 30 min. The slides were then incubated in mouse monoclonal anti α-SMA primary antibody (Sigma) in 1% Bovine Serum Albumin (BSA)/PBS (Sigma) for one hour. The sections were saturated in biotinylated horse anti-mouse IgG (Vector laboratories) in 1% BSA/PBS, and 2% normal horse serum (Vector laboratories) for 30 min. Avidin-D Texas red (Vector Laboratories) fluorochrome was applied to the sections after the secondary antibody was washed off. The sections were then counter stained with 0.25 μg ml−1 4′, 6-Diamidino-2-phenylindole (DAPI; Sigma), coverslipped and stored at 4°C. Slides were subsequently imaged at 200× using a TR-FITC-DAPI triple filter under a mercury lamp.
Image Analysis
H & E images were analyzed by measuring the fibrosa: spongiosa length ratio, and the fibrosa: ventricularis length ratio. α-SMA immunopositive cells appear red with cell nuclei counterstained blue, while the color of collagen fibers changes from green to yellow to orange to red as the fiber thickness (and consequently maturity) increases. The proportion of α-SMA positive cells were therefore assessed by using the Image J program (NIH, Bethesda, MD) to determine the relative area coverage of α-SMA positive red staining compared to the area coverage of entire leaflet section. The relative proportion of the different maturity of the collagen fibers in the leaflet section were calculated using published methods.2 Basically, picrosirius red images were first imported into Matlab 7.01 (The Mathworks, Natick, MA) and converted into their hue, saturation and value components. Only the hue components were retained, and a histogram of hue frequency was obtained from the 8-bit hue images that contained 256 possible colors. The colors were defined as follows: red 2–9 and 230–256; orange 10–38; yellow 39–51; and green 52–128. All other hue values corresponded to interstitial space and were confirmed by visual inspection. The number of pixels within each hue range were determined and expressed as a percentage of the total number of collagen pixels and plotted.
Immunoblotting Analysis
The rest of the leaflet sections were processed for analysis by immunoblotting. Leaflets were homogenized and sonicated in ice-cold lysis buffer (in mmol l−1: Tris–HCl 20 (pH 7.5), EGTA 5, NaCl 150, glycerol-phosphate 20, NaF 10, sodium orthovanadate 1, 1% Triton X−100, 0.1% Tween-20 and protease inhibitors) to extract proteins from the tissue, and then centrifuged at 14,000 rpm to pellet cellular debris. The supernatant was assayed for protein concentration using the DC Protein Assay (Bio-Rad, Hercules, CA) and equal aliquots of tissue lysates were resolved on a 10 % SDS-PAGE gel and subsequently blotted to a polyvinyldifluoride (PVDF) membrane (Millipore, Billerica, MA). Membranes were incubated with a mouse anti α-SMA primary antibody (Sigma) at 4°C overnight (1:1000), and then with a goat anti-mouse alkaline phosphatase-conjugated secondary antibody (Bio-Rad). CDP-Star reagent (NE Biolabs, Ipswich, MA) was added and chemiluminescence of the resulting product was detected using photographic paper. GAPDH antibody (Santa Cruz Biotech, Santa Cruz, CA) was used as a loading control (1:5000). Pixel densities of α-SMA immunoreactive bands were then analyzed using the Image J program and normalized by the intensities of the GAPDH immunoreactive bands.
Analysis of Tissue Culture Medium
As a follow-up to determine if sGAGs were leaching out from the leaflet in the DMEM, six samples of the DMEM were analyzed before and after the experiment. The BlyscanTM sGAG assay kit (Biocolor) used in the ECM analysis was used to detect sGAGs in the medium.
Statistical Analysis
The data for collagen, sGAG and elastin contents were expressed as mean plus one standard error of the mean. A total of 27 leaflets were analyzed for the ECM assays, 8 leaflets for the histological and immunohistochemical studies, and 10 leaflets for the Western blotting studies. The quantitative data from the ECM assays, image analysis and immunoblotting were analyzed first using ANOVA followed by pairwise comparisons with the Tukey post-hoc test. A p-value of less than 0.05 was used as a measure of statistical significance. When only two groups were being compared, data were analyzed using student t-tests with a p-value of less than 0.05 indicating statistical significance. All statistical analyses were performed using Minitab (Minitab R14, Minitab Inc. State College, PA).
Results
Validation of Stretch Bioreactor
The stretch bioreactor produced a uniform output strain across the leaflet of 14.80 ± 0.53% for 15% input strain, and 25.48 ± 0.53% for 25% input strain. Performing a student t-test between input and output signals revealed no significant difference (p > 0.05, n = 16), indicating that the bioreactor could accurately and uniformly reproduce the specified input strain across the entire leaflet section.
The peak steady-state wall shear stress experienced by the leaflet as calculated from CFD was 2.5 dyn cm−2. This is small compared to the peak 79 dyn cm−2 experienced by the leaflet under physiological flow conditions during systole as measured in our laboratory,28 and it can be assumed that stretch is the dominant force acting on the leaflet.
Extracellular Matrix Content1
A significant increase in collagen content was observed in stretched leaflets when compared to fresh and static control leaflets (p < 0.05, n = 27). Stretched leaflets showed a 90% and 161.5% increase in collagen content in comparison to fresh and static controls, respectively. There was no significant difference in collagen content between fresh and static control leaflets (p > 0.05, n = 27). sGAG content was found to be decreased by 40% in stretched leaflets when compared to fresh valve leaflets (p < 0.05, n = 27), while sGAG content was comparable between stretched and static control leaflets (p > 0.05, n = 27). ANOVA showed that there was no significant difference (p > 0.05, n = 27) between treatment groups for elastin content. The results for the ECM assays are graphically depicted in Fig. 3.
Figure 3.Cyclic stretch increased collagen, decreased sGAG, while not affecting elastin contents in aortic valve leaflets. The leaflets were exposed to physiologic circumferential cyclic stretch (15% at heart rate of 70 bpm) or static control conditions for 48 h. The amounts of collagen, sGAG and elastin in the leaflets exposed to static or stretching were compared to those of fresh leaflets. The data were normalized by tissue dry weight and expressed as a mean value plus one standard error of the mean (* p < 0.05, n = 27).
Analysis of the conditioned medium at the end of experimental duration revealed a statistically significant increase (p < 0.05, n = 6) in sGAG secreted in to the media (5.83 ± 0.56 μg ml−1 of DMEM) compared to the fresh media (1.08 ± 0.30).
Cell Morphology and Phenotype
H & E staining (Fig. 4a) of the leaflets showed preserved three-layered leaflet architecture in stretched leaflets consistent with fresh and static control leaflets. The cell nuclei were found to be intact with no tears in the tissue section. Image analysis showed no significant difference (p > 0.05, n = 8) in the fibrosa-spongiosa length ratio as well as the fibrosa-ventricularis length ratio (Fig. 5a) indicating that there was no gross damage to leaflets structure.
Figure 4.(a) Cyclic stretch did not damage tissue structure and morphology. H & E stained images of fresh, static and stretched aortic valve leaflets are depicted here. Cytoplasm was stained pink and cell nuclei were stained blue. The representative images show the three-layered morphology of the leaflets was intact in all three groups (F – fibrosa, S – spongiosa, V – ventricularis). (b) Cyclic stretch maintained native collagen architecture. Picrosirius red images of fresh, static and stretched aortic valve leaflets are shown. Mature collagen fibers were stained orange-red. Layered collagen morphology of leaflets was observed and crimp was preserved in stretched leaflets (F – fibrosa). (c) Cyclic stretch increased α-smooth muscle actin (α-SMA)-positive cells in the ventricularis side of the aortic valve leaflets. Fresh, static and stretched leaflets were examined by α-SMA IHC. Actin was stained red and cell nuclei were counterstained blue. Increased expression of α-SMA was observed in stretched leaflets, and α-SMA was reduced in static leaflets (V – ventricularis).Figure 5.(a) Fibrosa-to-spongiosa ratio and fibrosa-to-ventricularis ratio remained unchanged. There was no significant difference (p > 0.05, n = 8) in the relative thicknesses of the fibrosa, spongiosa and ventricularis layers between fresh, static and stretched leaflets. (b) Static leaflets exhibited reduced levels of newly synthesized collagen fibers. Newly synthesized fiber proportion was reduced in static leaflets signifying reduced levels of collagen synthesis. Newly synthesized fiber proportion was comparable between fresh and stretched leaflets, but the proportion of mature fibers was greater in stretched leaflets. (c) Cyclic stretch increases α-smooth muscle actin in the aortic valve leaflets. α-SMA immunopositive staining coverage was increased in stretched leaflets and reduced in static leaflets when compared with fresh leaflets.
Picrosirius red staining (Fig. 4b) of the leaflets showed that collagen architecture was preserved in fresh, static as well as stretched leaflets. The layered structure of collagen along with the crimp in the collagen fibers could clearly be seen in the stretched leaflets, demonstrating that the stretching motion did not damage valvular collagen. These findings demonstrated that uniaxial cyclic stretch of the leaflets did not damage leaflet morphology and maintained native ECM structure and cellular composition.
Additionally, hue analysis of the picrosirius red images (Fig. 5b) revealed that the proportion of newly synthesized (green) fibers in statically incubated leaflet sections was significantly decreased by 36.5% (p < 0.05, n = 8) compared to fresh leaflets. There was no significant difference in green fibers (p > 0.05, n = 8) between fresh and stretched leaflets, however, mature (red) collagen fibers significantly increased by 101.4% in stretched leaflets compared to fresh leaflets.
The immunohistochemical study with α-SMA (Fig. 4c), a marker of myofibroblast-like or smooth muscle cell-like valvular interstitial cells2, revealed that cyclic stretch up-regulated the expression of α-SMA, while static incubation down-regulated the expression of α-SMA. This was corroborated by image analysis (Fig. 5c) of the images, which showed significantly increased (p < 0.05, n = 8) α-SMA immunopositive staining in stretched leaflets compared to fresh leaflets, and significantly reduced (p < 0.05, n = 8) α-SMA staining in static leaflets compared to fresh leaflets. Another interesting observation was that the α-SMA positive cells in the stretched leaflets were predominantly found in the ventricularis region of the aortic valve leaflet while there was little or no α-SMA expression in the fibrosa. In contrast, α-SMA positive cells were randomly distributed in all the three regions of the fresh and static leaflets.
The above finding was further reinforced by the Western blotting results (Fig. 6). There was a 54.5% increase (p < 0.05, n = 10) in α-SMA content of stretched leaflets and 34.9% decrease (p < 0.05, n = 10) in statically incubated leaflets when compared to fresh leaflets. These results together suggest that cyclic stretching induces phenotypic change of valve interstitial cells toward myofibroblast-like cells in a side-dependent manner.
Figure 6.Cyclic stretch increased α-smooth muscle actin in the aortic valve leaflets. Fresh, static and stretched aortic valve leaflets were analyzed by Western blot with α-SMA antibody and analyzed by densitometry. When compared to fresh leaflets, α-SMA expression decreased in static leaflets, but increased in stretched leaflets (*p < 0.05, n = 10). Anti-GAPDH antibody was used as a loading control.
Discussion
Effect of Cyclic Stretch on ECM Components
The current study investigated the effects of isolated circumferential cyclic stretch on the biological responses of aortic valve leaflets using a sterile ex vivo stretch bioreactor. Initial validation of the bioreactor showed that strains were uniform across the leaflet section. It was also validated that there was negligible wall shear stress on the leaflet, and therefore cyclic stretch was the only force acting on the leaflet.
It was revealed that the collagen content of the aortic valve leaflets stretched to 15% was increased when compared to fresh and static control leaflets, while sGAG content was decreased in stretched leaflets compared to fresh leaflets. Furthermore, elastin content was comparable between the stretched and fresh leaflets. This suggests that exposure to isolated effects of circumferential cyclic stretch at physiological levels has altered the extracellular matrix composition, consequently, altering the valve remodeling.
Collagen fibers, densely populated in parallel alignment along the circumferential direction of the leaflet, form the load bearing structure of the leaflet during each cardiac cycle.18,26 The increase in collagen suggests that the leaflets adapt to altered mechanical loading due to the effects of isolated cyclic stretch, by either increasing synthesis, or decreasing degradation of collagen. The image analysis of the picrosirius red staining (Fig. 5b) showed comparable proportions of newly synthesized fibers, and hence collagen synthesis in both fresh and stretched leaflets. The increased proportion of mature (red) fibers in stretched leaflets therefore leads us to believe that it is the reduced degradation of collagen that causes the overall total increase in collagen content. The preserved crimp in collagen fibers in the stretched leaflet, as evidenced by picrosirius red staining also implies that the native structure of collagen was not damaged by the stretching motion.
This data is in agreement with a study conducted by Ku et al.14 where porcine aortic valve interstitial cells were cultured on a collagen substrate and stretched 48 h. For cells subjected to 14% cyclic stretch, collagen incorporation in the cell layer was approximately 170% of unstretched cells. No significant changes were observed at time points less than 48 h. This provides further evidence that 48 h of culture is a necessary and sufficient time point to observe changes in the valvular ECM.
The semi-fluidic nature of the sGAG in the spongiosa layer of the leaflet gives the leaflet considerable plasticity.26 It is therefore believed that the purpose of sGAG might be (i) to help reduce leaflet stresses by lubricating the relative motion of the other two layers as the leaflet deforms, and (ii) to dampen compressive forces during diastole.7 The absence of compressive stresses in this study, with the stretch being carried out at atmospheric pressure, might therefore explain the reduced levels of sGAG observed in the stretched leaflet compared to fresh leaflets. Additionally, analysis of the DMEM at the end of experimental duration showed a statistically significant increase in sGAG content in the media. This suggests that cyclic stretching of the aortic valve leaflets resulted in secreting or leaching of the sGAG from the leaflets into the media, as the various layers of the leaflet rub against each other. A study conducted by Grande-Allen et al.11 also showed a loss of GAG from explanted porcine bioprosthetic valves due to leaching even under normal storage conditions. This suggests that there is a high propensity for sGAGs to be forced out of the native aortic valve either during static incubation or leaflet motion. In contrast, aortic valve leaflets when exposed to normal physiological forces undergo constant sGAG renewal by the interstitial cells.13
In the ventricularis layer of the leaflet, the major extracellular component is elastin in the form of fibers that are oriented primarily along the radial direction. Their primary role of elastin fibers is to maintain a circumferential collagen fiber orientation and recoil the collagen fibers to their initial state after the external load has been released.27 Since the valve leaflets were cyclically stretched along the circumferential direction at physiological rates in the current study, it is possible that the elastin fibers were not “activated” beyond their normal levels and consequently additional synthesis of elastin was not required, explaining why elastin content remained unchanged between fresh and stretched leaflets.
The above findings reinforce the fact that valve cells require mechanical stimulation to maintain a balance between synthesis and degradation of ECM components. This occurred without undue damage to the normal tri-layered structure of the aortic valve leaflet as the relative thicknesses of the fibrosa, spongiosa, and ventricularis were maintained (Fig. 5a).
Effect on Cell Phenotype
Valvular interstitial cells are mainly comprised of fibroblasts, myofibroblasts, and smooth muscle cells that exhibit different phenotypes depending on the microenvironment surrounding the cells. Expression of α-SMA was associated with the contractile phenotype of smooth muscle cells24 and myofibroblast-like cells,2 and hence was chosen as a marker for examining the interstitial cell phenotype in the present study. Studies have shown that the expression of α-SMA was decreased by changes in culture conditions and mechanical and biochemical stimulation in cultured vascular cells.21,22 Studies in our laboratory have shown that α-SMA expression decreased in response to isolated effects of pressure and shear forces.13,29,31 Similar behavior was observed in valvular interstitial cells in 3D static gel culture.5
The current study revealed via IHC (Fig. 5c) and Western blotting (Fig. 6) that circumferential cyclic stretch increases the expression of α-SMA in aortic valve leaflets. It is therefore speculated that cyclic stretch is a regulator of the contractile and fibrotic phenotypes of aortic valve interstitial cells. A reversible equilibrium between F-actin depolymerization and G-actin polymerization exists in the cells of aortic valves. The contractile motion of the leaflets due to stretch may have caused a shift in this equilibrium towards polymerization of G-actin to form F-actin fibrils, which is detected as α-SMA immunopositive cells.
These findings also lead to the possibility that in the normal hemodynamic environment, the up-regulating effect of stretch on α-SMA expression may be counterbalanced by the down-regulating effects on α-SMA expression of pressure and shear stress. This provides an additional possible explanation for the fact that aortic valves cultured in normal hemodynamic forces maintain a constant α-SMA expression.13
Another interesting observation was that α-SMA immunopositive cells occurred preferentially in the ventricularis of the stretched leaflet. This is in contrast to fresh and static leaflets which exhibited an apparent random distribution of α-SMA through all the three layers of the valve leaflet. This may provide insights into specific regions or “hot spots” on the aortic valve leaflet where there is a focal response to mechanical forces. In a study by Simmons et al.,20 spatial heterogeneity of aortic valve endothelial phenotypes was speculated to contribute to the focal susceptibility for calcific aortic valve lesion development, implicating the endothelium as a regulator of valvular calcification. The current study shows the potential focal and side-specific response of the interstitial cells of the aortic valve to mechanical forces. In addition, α-SMA is a known marker for mesenchymal cells,3, 9 and the expression of α-SMA could also indicate regions of endothelial cell to mesenchymal cell transdifferentiation. It is clear that further side-specific study is needed in this area as it may provide insights into how valves sense and transmit mechanical forces.
Conclusion
The current study demonstrated that circumferential cyclic stretch altered the extracellular matrix remodeling activity of native porcine aortic valve leaflets. These results combined with results from previous ex vivo studies show that the changes in aortic valve leaflet biology depend intimately on the influence of local hemodynamic environmental factors. Further research is needed to understand the mechanistic pathways behind these biological changes. The knowledge gleaned from such studies will ultimately allow for elucidation of mechanisms of aortic valve pathogenesis leading to valve failure.

aortic valve leaflets
circumferential stretch
contractile phenotype
uniaxial stretch bioreactor