Node negative colorectal cancer patients: assesment of high-risk features on recurrence.

BACKGROUND: The introduction of population-based screening programs for colorectal cancer (CRC) results in less patients with advanced disease. There is an increase in the amount of node negative CRC, which makes adequate risk stratification for this particular group of patients necessary. The addition of more risk factors to the conventional histological high-risk factors is investigated in this retrospective study. PATIENTS AND METHODS: A cohort of 227 node negative (stage I and II) CRC patients who were not treated with adjuvant chemotherapy were selected from two previously conducted cohort studies. Detailed histopathological examination was performed by two independent observers and molecular background (BRAF/RAS mutations, microsatellite status (MSI)) was studied. Univariate analyses were used to analyse differences in histological and mutational characteristics between patients with and without recurrence. P-values below 0.05 were considered statistically significant. RESULTS: Poorly differentiated histology (p:0.002), BRAF mutation (p:0.002) and MSI status (p:0.006) were found significant relevant risk factors that were related to recurrent disease. Poorly differentiated histology was associated with intermediate/high tumor budding (TB) (p:0.001), a BRAF mutation (p:0.001) and MSI status (p:0.001). A combination of all three features (poorly differentiated histology, BRAF and MSI) was more often present in the recurrence group. CONCLUSIONS: Recurrence in node negative CRC patients could be better predicted when molecular features such as, BRAF mutation and MSI status are incorporated into a model with poorly differentiated CRC. Therefore, these features might help in the selection of patients who possibly will benefit from adjuvant treatment.

Convective-Dispersion Modeling in 3D Contrast-Ultrasound Imaging for the Localization of Prostate Cancer.

Despite being the solid tumor with the highest incidence in western men, prostate cancer (PCa) still lacks reliable imaging solutions that can overcome the need for systematic biopsies. Dynamic contrast-enhanced ultrasound imaging (DCE-US) allows us to quantitatively characterize the vascular bed in the prostate, due to its ability to visualize an intravenously administered bolus of contrast agents. Previous research has demonstrated that DCE-US parameters related to the vascular architecture are useful markers for the localization of PCa lesions. In this paper, we propose a novel method to assess the convective dispersion (D) and velocity (v) of the contrast bolus spreading through the prostate from three-dimensional (3D) DCE-US recordings. By assuming that D and v are locally constant, we solve the convective-dispersion equation by minimizing the corresponding regularized least-squares problem. 3D multiparametric maps of D and v were compared with 3D histopathology retrieved from the radical prostatectomy specimens of six patients. With a pixel-wise area under the receiver operating characteristic curve of 0.72 and 0.80, respectively, the method shows diagnostic value for the localization of PCa.

Prognostic implications of occult nodal tumour cells in stage I and II colon cancer: The correlation between micrometastasis and disease recurrence.

INTRODUCTION: Occult nodal tumour cells should be categorised as micrometastasis (MMs) and isolated tumour cells (ITCs). A recent meta-analysis demonstrated that MMs, but not ITCs, are prognostic for disease recurrence in patients with stage I/II colon cancer. AIMS & METHODS: The objective of this retrospective multicenter study was to correlate MMs and ITCs to characteristics of the primary tumour, and to determine their prognostic value in patients with stage I/II colon cancer. RESULTS: One hundred ninety two patients were included in the study with a median follow up of 46 month (IQR 33-81 months). MMs were found in eight patients (4.2%), ITCs in 37 (19.3%) and occult tumour cells were absent in 147 patients (76.6%). Between these groups, tumour differentiation and venous or lymphatic invasion was equally distributed. Advanced stage (pT3/pT4) was found in 66.0% of patients without occult tumour cells (97/147), 72.9% of patients with ITCs (27/37), and 100% in patients with MMs (8/8), although this was a non-significant trend. Patients with MMs showed a significantly reduced 3 year-disease free survival compared to patients with ITCs or patients without occult tumour cells (75.0% versus 88.0% and 94.8%, respectively, p = 0.005). When adjusted for T-stage, MMs independently predicted recurrence of cancer (OR 7.6 95% CI 1.5-37.4, p = 0.012). CONCLUSION: In this study, the incidence of MMs and ITCs in patients with stage I/II colon cancer was 4.2% and 19.3%, respectively. MMs were associated with an reduced 3 year disease free survival rate, but ITCs were not.

High-risk human papillomavirus detection in self-sampling compared to physician-taken smear in a responder population of the Dutch cervical screening: Results of the VERA study.

In 2017 the cervical cancer screening program in The Netherlands will be revised. Cervical smears will primarily be tested for the presence of high-risk human papillomavirus (hrHPV) instead of cytology, and vaginal self-sampling will be offered to non-responders. This includes a potential risk that part of the women who would otherwise opt for a cervical smear will wait for self-sampling. However, self-sampling for hrHPV in a responder population has never been studied yet. The aim of this study was to investigate the applicability and accuracy of self-sampling in detecting hrHPV in a screening responder population. A total of 2049 women, aged 30-60years, participating in the screening program in The Netherlands were included from April 2013 to May 2015. After they had their cervical smear taken, women self-collected a cervicovaginal sample with a brush-based device, the Evalyn Brush. Both the cervical smear and self-sample specimen were tested with the COBAS 4800 HPV platform. The hrHPV prevalence was 8.0% (95% CI 6.9-9.2) among the physician-taken samples, and 10.0% (95% CI 8.7-11.3) among the self-samples. There was 96.8% (95% CI 96.0-97.5) concordance of hrHPV prevalence between self-samples and physician-taken samples. Women in our study evaluated self-sampling as convenient (97.1%), user-friendly (98.5%), and 62.8% preferred self-sampling over a physician-taken sampling for the next screening round. In conclusion, self-sampling showed high concordance with physician-taken sampling for hrHPV detection in a responder screening population and highly acceptable to women. Implementation of HPV-self-sampling for the responder population as a primary screening tool may be considered.

Epidural analgesia associated with better survival in colon cancer.

PURPOSE: Surgery remains the mainstay of treatment for potentially curable colon cancer. Otherwise, the surgical stress response might increase the likelihood of cancer dissemination during and after cancer surgery. There is growing evidence that the type of anaesthesia during cancer surgery plays a role in the metastatic process. Therefore, we assessed if the method of anaesthesia is associated with long-term survival after colon cancer surgery. METHOD: A retrospective single-centre study was conducted including 588 patients who underwent colorectal cancer surgery, TNM stage I-IV, in the Jeroen Bosch Hospital between 1995 and 2003. The Cox proportional hazard model was used for statistical analysis. Adjustments were made for age, sex, comorbidity, TNM stage, chemotherapy, emergency surgery status and year of incidence. RESULTS: Of the 588 primary colon cancer patients with a median age of 70 years, 399 (68 %) patients underwent colon surgery with epidural anaesthesia, whilst 189 (32 %) patients were operated without epidural anaesthesia. Five-year survival for patients not receiving epidural analgesia was 42 % versus 51 % for patients receiving epidural analgesia (p = 0.03). This effect remained after adjustment for relevant patient, tumour, and treatment characteristics (hazard ratio (HR) 1.30 (95 % confidence interval (CI) 1.05-1.59), p = 0.01). Subgroup analysis in patients of 80 years and older (n = 100) showed also a better overall survival after receiving epidural analgesia (HR 1.74 (95 % CI 1.11-2.72), p = 0.01). CONCLUSION: Epidural analgesia during colon cancer surgery was associated with a better overall survival. Prospective trials evaluating the effects of locoregional analgesia on colon cancer recurrence are warranted.

Electromagnetic fields do not affect bone micro-architecture in osteoporotic rats.

OBJECTIVES: Electromagnetic fields (EMF) are widely used in musculoskeletal disorders. There are indications that EMF might also be effective in the treatment of osteoporosis. To justify clinical follow-up experiments, we examined the effects of EMF on bone micro-architectural changes in osteoporotic and healthy rats. Moreover, we tested the effects of EMF on fracture healing. METHODS: EMF (20 Gauss) was examined in rats (aged 20 weeks), which underwent an ovariectomy (OVX; n = 8) or sham-ovariectomy (sham-OVX; n = 8). As a putative positive control, all rats received bilateral fibular osteotomies to examine the effects on fracture healing. Treatment was applied to one proximal lower leg (three hours a day, five days a week); the lower leg was not treated and served as a control. Bone architectural changes of the proximal tibia and bone formation around the osteotomy were evaluated using in vivo microCT scans at start of treatment and after three and six weeks. RESULTS: In both OVX and sham-OVX groups, EMF did not result in cancellous or cortical bone changes during follow-up. Moreover, EMF did not affect the amount of mineralised callus volume around the fibular osteotomy. CONCLUSIONS: In this study we were unable to reproduce the strong beneficial findings reported by others. This might indicate that EMF treatment is very sensitive to the specific set-up, which would be a serious hindrance for clinical use. No evidence was found that EMF treatment can influence bone mass for the benefit of osteoporotic patients. Cite this article: Bone Joint Res 2014;3:230-5.

The diagnostic value of one-step nucleic acid amplification (OSNA) for sentinel lymph nodes in colon cancer patients.

BACKGROUND: Lymph node status in colon cancer is critical for prognosis estimation and treatment allocation. The purpose of this study was to compare the performance of one-step nucleic acid amplification (OSNA) through detection of cytokeratin 19 mRNA levels with routine pathological examination (RP) and multilevel fine pathological examination (FP) in sentinel lymph nodes (SLN), detected using the ex vivo SLN mapping (SLNM) procedure, in presurgically defined nonmetastatic colon cancer patients. METHODS: In this prospective study, 325 SLNs of 128 patients from the Jeroen Bosch Hospital in 's-Hertogenbosch and the Leiden University Medical Center were investigated by RP (H&E), FP (H&E and Keratin Pan immunohistochemical staining), and OSNA. The SLNs were harvested by the SLNM procedure, using Patent blue or Indocyanine green. SLNs were divided and separate parts were used for RP, FP, and the OSNA assay. RESULTS: The diagnostic value of OSNA was 82.1 and 100 % for both FP and combined method (OSNA and FP) compared with RP. An upstaging rate of 20.2 % was obtained with the use of OSNA only and 36.4 % with the use of FP only. An upstaging rate of 46.5 % was obtained by combining the two methods together. CONCLUSIONS: OSNA and FP appeared to be promising tools for the detection of lymph node micro- and macrometastases in SLNs after SLNM. The performances of OSNA and FP in this study were superior to RP. Because OSNA allows analysis of the whole lymph node, sampling bias can be avoided. OSNA therefore may improve tumor staging.

Lymph node metastasis after a soft tissue sarcoma of the leg: a case report and a review of the literature.

Introduction. Soft tissue sarcomas (STSs) represent 1 percent of all adult malignancies and sarcomas only rarely spread to the regional lymph nodes. Case Presentation. We present a case of a woman with a dermatofibrosarcoma protuberans and a sarcoma not therwise specified of the lower extremity. The patient had no distant metastasis during follow-up, but did develop a regional lymph nodemetastasis (RLNM) in the groin. We reviewed the literature about RLNM in STSs. Discussion. Reviewing the literature we see that within specific histological types RLNM occurs as often as distant metastasis. Furthermore RLNM occurs in over 10% for specific histological types and in 24% of all patients with a soft tissue sarcoma of the lower extremity. Except for radical lymphadenectomy with a 5-year survival rate of 46% there is no appropriate treatment. Conclusion. The risk for a RLNM in certain histological types and anatomical locations might transcend the risk for a distant lung metastasis.

Ultrasound-guided radiofrequency ablation of early breast cancer in a resection specimen: lessons for further research.

PURPOSE: To assess the feasibility and effectiveness of radiofrequency ablation (RFA) in breast cancer, using different histopathologic staining methods to evaluate tissue viability. MATERIALS AND METHODS: In twenty patients with unifocal small (≤1, 5 cm) invasive ductal carcinoma, ultrasound-guided RFA was performed immediately after surgery. Cell viability was assessed using cytokeratin 8 (CK 8) and nicotinamide adenine dinucleotide diaphorase (NADHD) in addition to hematoxylin-eosin (HE). RESULTS: At histopathological examination, ex vivo RFA resulted in complete cell death of the target lesion in 17/20 patients. In two cases viable ductal carcinoma in situ (DCIS) was found just outside the completely ablated lesion. CONCLUSION: RFA of small invasive breast cancer seems to be a feasible treatment option. Both NADHD and CK 8 demonstrate a clear and comparable demarcation between viable and non-viable tissue. A high level of accuracy is required in proper positioning of the needle electrode and a "hot retraction" is mandatory.

The number of high-risk factors is related to outcome in stage II colonic cancer patients.

BACKGROUND: A subgroup of stage II colonic cancer patients are considered to be at high-risk for recurrent/metastatic disease based on 1) tumour obstruction/perforation 2) <10 lymph nodes 3) T4 lesions and 4) lymphangio-invasion. Their prognosis is regarded as comparable to stage III (T1-4N+M0) colonic cancer and it is therefore strongly advised to treat them with adjuvant chemotherapy. The purpose of this study was i) to determine the magnitude of prognostic significance of the conventional high-risk factors and ii) to determine whether the number of high-risk factors influences outcome. METHODS: We retrospectively analyzed 212 stage II colonic cancer patients undergoing surgery between January 2002 and December 2008. No adjuvant chemotherapy was given. Survival analyses were performed. RESULTS: 154/212 (73%) patients were considered to be high-risk patients based on conventional high-risk factors. 58 patients did not meet any high-risk factor, 125 patients met 1 high-risk factor and 29 patients met ≥2 high-risk factors. Median follow up was 40 months. Multivariate analysis identified four independent risk factors for recurrent/metastatic disease: age, obstruction, perforation and lymphangio-invasion. The three-year-DFS-rates for the low-risk group, the high-risk group with 1 high-risk factor and the high-risk group with ≥2 high-risk criteria are 90.4%, 87.6% and 75.9% respectively. Patients meeting ≥2 conventional high-risk criteria had a significantly worse three-year disease free survival (p < 0.002). CONCLUSIONS: Four independent high-risk factors were identified. The number of high-risk factors does influence outcome. More attention should be given to the definition and treatment of high-risk stage II colonic cancer patients.

Unfocused extracorporeal shock waves induce anabolic effects in rat bone.

BACKGROUND: Extracorporeal shock waves are known to stimulate the differentiation of mesenchymal stem cells toward osteoprogenitors and induce the expression of osteogenic-related growth hormones. The aim of this study was to investigate if and how extracorporeal shock waves affected new bone formation, bone microarchitecture, and the mechanical properties of bone in a healthy rat model, in order to evaluate whether extracorporeal shock wave therapy might be a potential treatment for osteoporosis. METHODS: Thirteen rats received 1000 electrohydraulically generated unfocused extracorporeal shock waves to the right tibia. The contralateral, left tibia was not treated and served as a control. At two, seven, twenty-one, and forty-nine days after administration of the shock waves, in vivo single-photon-emission computed tomography (SPECT) scanning was performed to measure new bone formation on the basis of uptake of technetium-labeled methylene diphosphonate ((99m)Tc-MDP) (n = 6). Prior to and forty-nine days after the extracorporeal shock wave therapy, micro-computed tomography (micro-CT) scans were made to examine the architectural bone changes. In addition, mechanical testing, microcrack, and histological analyses were performed. RESULTS: Extracorporeal shock waves induced a strong increase in (99m)Tc-MDP uptake in the treated tibia compared with the uptake in the untreated, control tibia. Micro-CT analysis showed that extracorporeal shock waves stimulated increases in both trabecular and cortical volume, which resulted in higher bone stiffness compared with that of the control tibiae. Histological analysis showed intramedullary soft-tissue damage and de novo bone with active osteoblasts and osteoid in the bone marrow of the legs treated with extracorporeal shock waves. Microcrack analysis showed no differences between the treated and control legs. CONCLUSIONS: This study shows that a single treatment with extracorporeal shock waves induces anabolic effects in both cancellous and cortical bone, leading to improved biomechanical properties. Furthermore, treatment with extracorporeal shock waves results in transient damage to the bone marrow, which might be related to the anabolic effects. After further examination and optimization, unfocused extracorporeal shock waves might enable local treatment of skeletal sites susceptible to fracture.

Use of cytokeratin 8 immunohistochemistry for assessing cell death after radiofrequency ablation of breast cancers.

The effects of minimally invasive therapies such as radiofrequency ablation (RFA) and laser induced thermal therapy on breast carcinoma lesions usually is assessed by NADH diaphorase enzyme histochemistry for cell viability. NADH staining requires frozen material, however, with its associated poor morphology. We aimed to validate cytokeratin 8 (CK 8) immunohistochemistry as an alternative that works on paraffin sections. RFA was performed ex vivo on 20 breast resections after surgery and in vivo in eight patients who underwent general anesthesia followed by immediate resection. After treatment, specimens were lamellated and the tumors were divided into two equal parts. One part was fixed in neutral buffered formaldehyde for routine histopathological evaluation using hematoxylin and eosin (H & E) staining and CK 8 immunostaining. The other section was snap frozen and stored at -80° C for staining with NADH diaphorase. Both NADH diaphorase and CK 8 immunostaining demonstrated a clear and comparable demarcation between viable and nonviable tissues. The morphology of the CK 8 immunostained slides was much better, and fatty tissues could be judged readily by contrast to the NADH stained frozen sections, which had poor morphology and whose fatty parts were difficult to interpret. CK 8 immunohistochemistry seems to be well suited for assessing cell viability in breast tissue and for assessing the effects of RFA for breast cancer treatment. Because it can be applied to paraffin fixed material, it provides much better morphology than NADH staining and also can be applied to fatty tissues that usually are difficult to work up for frozen sections. Therefore, CK 8 immunohistochemistry may be preferred over NADH diaphorase staining for daily pathology practice for assessing the viability of breast carcinoma cells after RFA treatment.

Influence of the positioning of a cementless glenoid prosthesis on its interface micromotions.

The positioning of the glenoid component in total shoulder arthroplasty is complicated by the limited view during operation. Malalignment and/or motion of the glenoid component with respect to the bone can be a cause of, or contribute to, failure of the implant. The aim of this paper is to determine the effect of the positioning of a cementless glenoid component on the micromotions between the implant and the bone during normal loading after surgery. For this study a three-dimensional finite element model of a complete scapula with a cementless glenoid component was used. In total, eight positions of the upper arm in both abduction and anteflexion were chosen to represent the patient's arm movement postoperatively. A previously published musculoskeletal model was used to determine the joint and muscle forces on the scapula with implant in each arm position. Five different alignments of the glenoid component (neutral, anterior, inferior, posterior, and superior inclinations), two different implantation depths ('optimal' and 'deeper' implantations), and two bone qualities (healthy and rheumatoid arthritis (RA) bone) were considered. Inclinations of 10 degrees with respect to a neutral alignment did not affect the overall interface micromotions in the optimal implantation depth. However, when the implantation depth was 3 mm deeper, anterior and inferior inclinations were more favourable than a neutral alignment and other inclinations. Micromotions in RA bone were always larger than in healthy bone.

[An invasive process in the pancreas: sometimes lymphoma]. / Een ruimte-innemend proces in het pancreas: soms een lymfoom.

An invasive process in the pancreas was found in a 60-year-old woman and a 50-year-old man with abdominal symptoms. Generally, such findings turn out to be adenocarcinoma. However, these patients had lymphoma. Primary pancreatic lymphoma or localization of lymphoma in the pancreas are rare and chemotherapy may be curative. Therefore, obtaining tissue for histopathological confirmation of the diagnosis is very important. Both patients underwent chemotherapy. The first patient was in complete remission one month after the last chemotherapy cycle. In the second, the disease went into remission, but he suddenly died of sepsis after the fourth chemotherapy cycle.

The value of an artificial neural network in the decision-making for prostate biopsies.

PURPOSE: In majority of patients who are subjected to prostate biopsies, no prostate cancer (PCa) is found. It is important to prevent unnecessary biopsies since serious complications may occur. An artificial neural network (ANN) may be able to predict the risk of the presence of PCa. METHODS: Included were all patients, who underwent transrectal ultrasound-guided prostate biopsies between June 2006 and June 2007 with a total PSA (tPSA) level between 2 and 20 microg/l. The patients were divided into two groups according to their tPSA level (2-10 microg/l and 10-20 microg/l). The ANN Prostataclass of the Universitätsklinikum Charité in Berlin was used. The predictions of the ANN were compared to the pathology results of the biopsies. RESULTS: Overall 165 patients were included. PCa was diagnosed in 53 patients, whereas the ANN predicted "no risk" in 19 of these patients (36%). The ANN output receiver operator characteristic (ROC) plots for the range of tPSA 2-10 microg/l and tPSA 10-20 microg/l showed an area under the curve (AUC) of 63 and 88% for the initial biopsy group, versus 69 and 57%, respectively, for the repeat biopsy group. CONCLUSIONS: The ANN resulted in a false negative rate of 36%, missing PCa in 19 patients. For use in an outpatient-clinical setting, this ANN is insufficient to predict the risk of presence of PCa reliably.

Micro-metastases in stages I and II colon cancer are a predictor of the development of distant metastases and worse disease-free survival.

Approximately 30% of the patients with Dukes A/B colon carcinoma will develop loco-regional recurrence or distant metastases. The aim of this study was to evaluate if patients with micro-metastases are at higher risk for developing distant metastases and therefore a worse disease-free survival and overall survival. In the period January 2000-January 2002, 137 patients underwent curative surgery for colon cancer. When patients had a Dukes A/B colon carcinoma, additional staining and sectioning on the harvested lymph nodes were performed retrospectively. Lymph nodes were examined using 4 multilevel sections at 250-microm intervals and stained with Pan-Cytokeratin. There were 11 patients with a Dukes A and 61 patients with a Dukes B colon carcinoma. Twenty-two patients developed metastases in time (group I) whereas 50 patients did not (group II). After additional staining and sectioning 41% of the patients of group I and 16% of the patients of group II showed micro-metastases (p<0.05). The 5-year overall survival rate in the group with micro-metastases was 62% against 79% in the group without micro-metastases. The disease-free survival (DFS) was 51% and 72% (p<0.05), respectively. Patients with micro-metastases develop significant more distant metastases in time and have a significant worse DFS.

Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis.

OBJECTIVE: To see how initial differences in subchondral bone phenotype influence the development of cartilage damage and changes in subchondral bone architecture in an osteoarthritis (OA)-induced mouse model. METHOD: Intra-articular collagenase injections (right knee joint) and saline controls (left knee joint) were applied in the knees of two mouse strains known to have either a low or a high bone mass phenotype: the low bone mass C57Bl/6 mice with a thin subchondral bone plate and high bone mass C3H/HeJ mice with a thick subchondral bone plate. The ages of the mice were 16 and 30 weeks, with n=8 per group. The collagenase injection induced an osteoarthritic phenotype that was evaluated 4 weeks later in the tibia using histological analyses and micro-computed tomography (micro-CT). RESULTS: Both strains developed cartilage damage in the collagenase-injected right knee joints to a comparable extent, however, the spatial distribution of cartilage damage differed significantly: C57Bl/6 mice had most damage at the postero-lateral side, whereas in C3H/HeJ mice the postero-medial region was the most affected. Spontaneous cartilage damage was found in the saline-injected left control knees of C57Bl/6 mice, but in C3H/HeJ mice spontaneous cartilage damage was virtually absent. In both strains the subchondral bone plate of collagenase-injected joints became thinner, independent of the site of cartilage damage. TRAP-positive osteoclasts were observed underneath the subchondral bone plate, in line with the observed decreased thickness. No link was found between subchondral bone plate thickness and cartilage damage in the collagenase-injected joints. The subchondral trabecular architecture only changed in the high bone mass C3H/HeJ mice, with thinning of trabeculae and increased trabecular spacing. CONCLUSION: Thinning of the subchondral bone plate was found as a common observation 4 weeks after OA had been induced in two strains of mice having either a high or low bone phenotype, but no relation was found with the amount of cartilage damage. In addition, this study shows that different strains of mice can react differently to instability-induced OA with respect to the spatial arrangement of cartilage damage and changes in subchondral trabecular structure.

In vitro expansion affects the response of chondrocytes to mechanical stimulation.

OBJECTIVE: Expansion of autologous chondrocytes is a common step in procedures for cartilage defect repair. Subsequent dedifferentiation can alter cellular response to mechanical loading, having major consequences for the cell's behavior in vivo after reimplantation. Therefore, we examined the response of primary and expanded human articular chondrocytes to mechanical loading. METHOD: Primary and expanded chondrocytes were stretched at either 0.5% or 3.0% at 0.5Hz, 2h per day, for 3 days. Gene expression levels of matrix components (aggrecan (AGC1), lubricin (PRG4), collagen type I (COL1), type II (COL2) and type X (COL10)) as well as matrix enzymes (matrix metalloproteinase 1 (MMP1), MMP3, MMP13) and SOX9 were compared to unstretched controls. To evaluate the effect of a chondrogenic environment on cellular response to stretch, redifferentiation medium was used on expanded cells. RESULTS: In primary chondrocytes, stretch led to mild decreases in AGC1, COL1 and COL10 gene expression (maximum of 3.8-fold) and an up-regulation of PRG4 (2.0-fold). In expanded chondrocytes, expression was down-regulated for AGC1 (up to 21-fold), PRG4 (up to 5.0-fold), COL1 (10-fold) and COL2 (2.9-fold). Also, expression was up-regulated for MMP1 (20-fold) and MMP3 (up to 4-fold), while MMP13 was down-regulated (2.8-fold). A chondrogenic environment appeared to temper effects of stretch. DISCUSSION: Our results show that expansion alters the response of human chondrocytes to stretch. Expanded chondrocytes greatly decrease gene expression of matrix constituents and increase expression of MMPs, whereas primary chondrocytes hardly respond. Our data could be a reference for optimization of cell sources or expansion protocols for reimplanted chondrocytes.

Contribution of collagen network features to functional properties of engineered cartilage.

BACKGROUND: Damage to articular cartilage is one of the features of osteoarthritis (OA). Cartilage damage is characterised by a net loss of collagen and proteoglycans. The collagen network is considered highly important for cartilage function but little is known about processes that control composition and function of the cartilage collagen network in cartilage tissue engineering. Therefore, our aim was to study the contribution of collagen amount and number of crosslinks on the functionality of newly formed matrix during cartilage repair. METHODS: Bovine articular chondrocytes were cultured in alginate beads. Collagen network formation was modulated using the crosslink inhibitor beta-aminopropionitrile (BAPN; 0.25mM). Constructs were cultured for 10 weeks with/without BAPN or for 5 weeks with BAPN followed by 5 weeks without. Collagen deposition, number of crosslinks and susceptibility to degradation by matrix metalloproteinase-1 (MMP-1) were examined. Mechanical properties of the constructs were determined by unconfined compression. RESULTS: BAPN for 5 weeks increased collagen deposition accompanied by increased construct stiffness, despite the absence of crosslinks. BAPN for 10 weeks further increased collagen amounts. Absence of collagen crosslinks did not affect stiffness but ability to hold water was lower and susceptibility to MMP-mediated degradation was increased. Removal of BAPN after 5 weeks increased collagen amounts, allowed crosslink formation and increased stiffness. DISCUSSION: This study demonstrates that both collagen amounts and its proper crosslinking are important for a functional cartilage matrix. Even in conditions with elevated collagen deposition, crosslinks are needed to provide matrix stiffness. Crosslinks also contribute to the ability to hold water and to the resistance against degradation by MMP-1.