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Significance The inadequate mechanical properties of engineered tissues have prevented related therapies from clinical translation. Collagen cross-links correlate with the mechanical integrity of tissues; however, addressing the weakness of neotissues through enhancing collagen cross-links has not received the attention it deserves. The present study demonstrates, both in vitro and in vivo, that improvements in the mechanical properties of native and engineered tissues can be attained using endogenous (hypoxia-mediated) and exogenous application of lysyl oxidase, which is the enzyme responsible for collagen cross-linking. By promoting an ∼16-fold increase in collagen cross-linking and, concomitantly, an approximately fivefold enhancement in the neotissue’s mechanical properties, this work creates new prospects for regenerative medicine. The methods developed here work across a spectrum of collagen-rich tissues and are clinically applicable. The inability to recapitulate native tissue biomechanics, especially tensile properties, hinders progress in regenerative medicine. To address this problem, strategies have focused on enhancing collagen production. However, manipulating collagen cross-links, ubiquitous throughout all tissues and conferring mechanical integrity, has been underinvestigated. A series of studies examined the effects of lysyl oxidase (LOX), the enzyme responsible for the formation of collagen cross-links. Hypoxia-induced endogenous LOX was applied in multiple musculoskeletal tissues (i.e., cartilage, meniscus, tendons, ligaments). Results of these studies showed that both native and engineered tissues are enhanced by invoking a mechanism of hypoxia-induced pyridinoline (PYR) cross-links via intermediaries like LOX. Hypoxia was shown to enhance PYR cross-linking 1.4- to 6.4-fold and, concomitantly, to increase the tensile properties of collagen-rich tissues 1.3- to 2.2-fold. Direct administration of exogenous LOX was applied in native cartilage and neocartilage generated using a scaffold-free, self-assembling process of primary chondrocytes. Exogenous LOX was found to enhance native tissue tensile properties 1.9-fold. LOX concentration- and time-dependent increases in PYR content (∼16-fold compared with controls) and tensile properties (approximately fivefold compared with controls) of neocartilage were also detected, resulting in properties on par with native tissue. Finally, in vivo subcutaneous implantation of LOX-treated neocartilage in nude mice promoted further maturation of the neotissue, enhancing tensile and PYR content approximately threefold and 14-fold, respectively, compared with in vitro controls. Collectively, these results provide the first report, to our knowledge, of endogenous (hypoxia-induced) and exogenous LOX applications for promoting collagen cross-linking and improving the tensile properties of a spectrum of native and engineered tissues both in vitro and in vivo.

Background Radiographic calcifications and cystic morphology are associated with higher and lower tumor grade, respectively, in pancreatic neuroendocrine tumors (PNETs). Whether calcifications and/or cystic morphology could be used preoperatively to predict post-resection survival in patients with PNETs remains elusive. Methods Patients undergoing curative-intent resection of well-differentiated PNETs from 2000 to 2017 at eight academic institutions participating in the US Neuroendocrine Tumor Study Group were identified. Preoperative cross-sectional imaging reports were reviewed to identify the presence of calcifications and of a cystic component occupying >50% of the total tumor area. Clinicopathologic characteristics and recurrence-free survival (RFS) were compared. Results Of 981 patients studied, 18% had calcifications and 17% had cystic tumors. Tumors with calcifications were more commonly associated with Ki-67 ≥3% (47% vs. 33%; p  = 0.029), lymph node metastasis (36% vs. 24%; p  = 0.011), and distant metastasis (13% vs. 4%; p  < 0.001). In contrast, cystic tumors were less commonly associated with lymph node metastasis (12% vs. 30%; p  < 0.001). Five-year RFS after resection was most favorable for cystic tumors without calcifications (91%), intermediate for solid tumors without calcifications (77%), and least favorable for any calcified PNET (solid 69%, cystic 67%; p  = 0.043). Calcifications remained an independent predictor of RFS on multivariable analysis ( p  = 0.043) controlling for nodal ( p  < 0.001) and distant metastasis ( p  = 0.001). Conclusions Easily detectable radiographic features, such as calcifications and cystic morphology, can be used preoperatively to stratify prognosis in patients with PNETs and possibly inform the decision to operate or not, as well as guide the extent of resection and potential use of neoadjuvant therapy.

Articular cartilage does not integrate due primarily to a scarcity of cross-links and viable cells at the interface. The objective of this study was to test the hypothesis that lysyl-oxidase, a metalloenzyme that forms collagen cross-links, would be effective in improving integration between native-to-native, as well as tissue engineered-to-native cartilage surfaces. To examine these hypotheses, engineered cartilage constructs, synthesized via the self-assembling process, as well as native cartilage, were implanted into native cartilage rings and treated with lysyl-oxidase for varying amounts of time. For both groups, lysyl-oxidase application resulted in greater apparent stiffness across the cartilage interface 2-2.2 times greater than control. The construct-to-native lysyl-oxidase group also exhibited a statistically significant increase in the apparent strength, here defined as the highest observed peak stress during tensile testing. Histology indicated a narrowing gap at the cartilage interface in lysyl-oxidase treated groups, though this alone is not sufficient to indicate annealing. However, when the morphological and mechanical data are taken together, the longer the duration of lysyl-oxidase treatment, the more integrated the interface appeared. Though further data are needed to confirm the mechanism of action, the enhancement of integration may be due to lysyl-oxidase-induced pyridinoline cross-links. This study demonstrates that lysyl-oxidase is a potent agent for enhancing integration between both native-to-native and native-to-engineered cartilages. The fact that interfacial strength increased manifold suggests that cross-linking agents should play a significant role in solving the difficult problem of cartilage integration. Future studies must examine dose, dosing regimen, and cellular responses to lysyl-oxidase to optimize its application.

Background Overall survival (OS) has traditionally been the primary end point in studies evaluating the clinical benefit of first-line chemotherapy in metastatic, locally advanced, or unresectable pancreatic cancer (MLAUPC). Given the prolonged follow-up assessment required to obtain OS and its potential to be confounded by second-line treatments, this study sought to determine whether progression-free survival (PFS), response rate (RR), or disease control rate (DCR) can serve as a reliable surrogate for OS. Methods A systematic review and meta-analysis was performed including all phase 3 clinical trials for MLAUPC, with gemcitabine as the control arm of the trial. The hazard ratios (HRs) for OS and PFS and odds ratios (ORs) for RR and DCR were recorded. A weighted Pearson correlation coefficient was estimated for the association between OS and the other outcomes. The primary analysis used a random effects weighting model, whereas the secondary analyses used a fixed effects- or sample size-weighted approach. Results For the study, 24 randomized controlled trials were identified. The Pearson correlation coefficient between OS and PFS was 0.86 (95% confidence interval [CI] 0.67–0.94; p  < 0.001). Sensitivity analysis of the studies with little to no crossover further showed a correlation coefficient of 0.91 (95% CI 0.76–0.97; p  < 0.001). The correlation coefficient between OS and RR was 0.45 (95% CI 0.07–0.72; p  = 0.02) and between OS and DCR was 0.74 (95% CI 0.38–0.90; p  < 0.001). Conclusions First-line chemotherapy trials for MLAUPC show a robust correlation between OS and PFS, affirming its role as a surrogate of OS.

Key Points The limited ability of articular cartilage to regenerate has prompted the development of cell-based tissue engineering techniques, such as autologous chondrocyte implantation The complexity of autologous chondrocyte implantation and contraindications in wide clinical application have driven the development of matrix-assisted chondrocyte implantation, which uses scaffolds to provide mechanical stability and support chondrogenesis To improve neotissue formation, graft maturation and biomechanical integrity, cells can be cultured in vitro before implantation on 3D matrices with exogenous stimuli, such as growth factors Biomaterials such as collagen type I and III membranes are also used in cell-free approaches to enhance cartilage's innate regenerative capacity by functioning as scaffolds for resident progenitor cells Scaffold-free, cell-based techniques to form biomimetic neotissues, which can avoid disadvantages of scaffold use such as limited biocompatibility and release of degradation byproducts, are now in development Despite promising results and advances in cartilage tissue engineering, translation into clinical practice has not yet been achieved, as many challenges remain to be resolved Although osteoarthritis can be slowed by biologic therapy, surgical interventions are necessary to recover cartilage function. In this Review of cartilage repair techniques, the authors outline how currently accepted methods can fall short in providing a long-term solution of hyaline cartilage regeneration and discuss preclinical and clinical trials of new cell scaffold-based, cell-free scaffold-based and scaffold-free procedures. Chondral and osteochondral lesions due to injury or other pathology commonly result in the development of osteoarthritis, eventually leading to progressive total joint destruction. Although current progress suggests that biologic agents can delay the advancement of deterioration, such drugs are incapable of promoting tissue restoration. The limited ability of articular cartilage to regenerate renders joint arthroplasty an unavoidable surgical intervention. This Review describes current, widely used clinical repair techniques for resurfacing articular cartilage defects; short-term and long-term clinical outcomes of these techniques are discussed. Also reviewed is a developmental pipeline of acellular and cellular regenerative products and techniques that could revolutionize joint care over the next decade by promoting the development of functional articular cartilage. Acellular products typically consist of collagen or hyaluronic-acid-based materials, whereas cellular techniques use either primary cells or stem cells, with or without scaffolds. Central to these efforts is the prominent role that tissue engineering has in translating biological technology into clinical products; therefore, concomitant regulatory processes are also discussed.

BackgroundSurgical resection of hepatic metastases remains the only potentially curative treatment option for patients with colorectal liver metastases (CRLM). Widely adopted prognostic tools may oversimplify the impact of model parameters relative to long-term outcomes.MethodsPatients with CRLM who underwent a hepatectomy between 2001 and 2018 were identified in an international, multi-institutional database. Bootstrap resampling methodology used in tandem with multivariable mixed-effects logistic regression analysis was applied to construct a prediction model that was validated and compared with scores proposed by Fong and Vauthey.ResultsAmong 1406 patients who underwent hepatic resection of CRLM, 842 (59.9%) had recurrence. The full model (based on age, sex, primary tumor location, T stage, receipt of chemotherapy before hepatectomy, lymph node metastases, number of metastatic lesions in the liver, size of the largest hepatic metastases, carcinoembryonic antigen [CEA] level and KRAS status) had good discriminative ability to predict 1-year (area under the receiver operating curve [AUC], 0.693; 95% confidence interval [CI], 0.684–0.704), 3-year (AUC, 0.669; 95% CI, 0.661–0.677), and 5-year (AUC, 0.669; 95% CI, 0.661–0.679) risk of recurrence. Studies analyzing validation cohorts demonstrated similar model performance, with excellent model accuracy. In contrast, the AUCs for the Fong and Vauthey scores to predict 1-year recurrence were only 0.527 (95% CI, 0.514–0.538) and 0.525 (95% CI, 0.514–0.533), respectively. Similar trends were noted for 3- and 5-year recurrence.ConclusionThe proposed clinical score, derived via machine learning, which included clinical characteristics and morphologic data, as well as information on KRAS status, accurately predicted recurrence after CRLM resection with good discrimination and prognostic ability.

Intro Chromogranin A (CgA) may be prognostic for patients with neuroendocrine tumors; however, the clinical utility of this test is unclear. Methods Patients undergoing resection for pancreatic neuroendocrine tumors (pNET) were selected from the eight institutions of the US Neuroendocrine Tumor Study Group database. Cox regression was used to identify pre-operative variables that predicted recurrence-free survival (RFS), and those with p  < 0.1 were included in a risk score. The risk score was tested in a unique subset of the overall cohort. Results In the entire cohort of 287 patients, median follow-up time was 37 months, and 5-year RFS was 73%. Cox regression analysis identified four variables for inclusion in the risk score: CgA > 5x ULN (HR 4.3, p  = 0.01), tumor grade 2/3 (HR 3.7, p  = 0.01), resection for recurrent disease (HR 6.2, p  < 0.01), and tumor size > 4 cm (HR 4.5, p  = 0.1). Each variable was assigned 1 point. Risk-score testing in the unique validation cohort of 63 patients revealed a 95% negative predictive value for recurrence in patients with zero points. Discussion This simple pre-operative risk scoring system resulted in a high degree of specificity for identifying patients at low-risk for tumor recurrence. This test can be utilized pre-operatively to aid informed decision-making.

Toward developing engineered cartilage for the treatment of cartilage defects, achieving relevant functional properties before implantation remains a significant challenge. Various chemical and mechanical stimuli have been used to enhance the functional properties of engineered musculoskeletal tissues. Recently, Ca2+-modulating agents have been used to enhance matrix synthesis and biomechanical properties of engineered cartilage. The objective of this study was to determine whether other known Ca2+ modulators, digoxin and adenosine triphosphate (ATP), can be employed as novel stimuli to increase collagen synthesis and functional properties of engineered cartilage. Neocartilage constructs were formed by scaffold-free self-assembling of primary bovine articular chondrocytes. Digoxin, ATP, or both agents were added to the culture medium for 1 h/day on days 10–14. After 4 weeks of culture, neocartilage properties were assessed for gross morphology, biochemical composition, and biomechanical properties. Digoxin and ATP were found to increase neocartilage collagen content by 52–110% over untreated controls, while maintaining proteoglycan content near native tissue values. Furthermore, digoxin and ATP increased the tensile modulus by 280% and 180%, respectively, while the application of both agents increased the modulus by 380%. The trends in tensile properties were found to correlate with the amount of collagen cross-linking. Live Ca2+ imaging experiments revealed that both digoxin and ATP were able to increase Ca2+ oscillations in monolayer-cultured chondrocytes. This study provides a novel approach toward directing neocartilage maturation and enhancing its functional properties using novel Ca2+ modulators.