Prognostic value of histopathological DCIS features in a large-scale international interrater reliability study.

PURPOSE: For optimal management of ductal carcinoma in situ (DCIS), reproducible histopathological assessment is essential to distinguish low-risk from high-risk DCIS. Therefore, we analyzed interrater reliability of histopathological DCIS features and assessed their associations with subsequent ipsilateral invasive breast cancer (iIBC) risk. METHODS: Using a case-cohort design, reliability was assessed in a population-based, nationwide cohort of 2767 women with screen-detected DCIS diagnosed between 1993 and 2004, treated by breast-conserving surgery with/without radiotherapy (BCS ± RT) using Krippendorff's alpha (KA) and Gwet's AC2 (GAC2). Thirty-eight raters scored histopathological DCIS features including grade (2-tiered and 3-tiered), growth pattern, mitotic activity, periductal fibrosis, and lymphocytic infiltrate in 342 women. Using majority opinion-based scores for each feature, their association with subsequent iIBC risk was assessed using Cox regression. RESULTS: Interrater reliability of grade using various classifications was fair to moderate, and only substantial for grade 1 versus 2 + 3 when using GAC2 (0.78). Reliability for growth pattern (KA 0.44, GAC2 0.78), calcifications (KA 0.49, GAC2 0.70) and necrosis (KA 0.47, GAC2 0.70) was moderate using KA and substantial using GAC2; for (type of) periductal fibrosis and lymphocytic infiltrate fair to moderate estimates were found and for mitotic activity reliability was substantial using GAC2 (0.70). Only in patients treated with BCS-RT, high mitotic activity was associated with a higher iIBC risk in univariable analysis (Hazard Ratio (HR) 2.53, 95% Confidence Interval (95% CI) 1.05-6.11); grade 3 versus 1 + 2 (HR 2.64, 95% CI 1.35-5.14) and a cribriform/solid versus flat epithelial atypia/clinging/(micro)papillary growth pattern (HR 3.70, 95% CI 1.34-10.23) were independently associated with a higher iIBC risk. CONCLUSIONS: Using majority opinion-based scores, DCIS grade, growth pattern, and mitotic activity are associated with iIBC risk in patients treated with BCS-RT, but interrater variability is substantial. Semi-quantitative grading, incorporating and separately evaluating nuclear pleomorphism, growth pattern, and mitotic activity, may improve the reliability and prognostic value of these features.

[A woman with vaginal tissue loss]. / Een vrouw met vaginaal weefselverlies.

A 44-year-old woman reported to the general practitioner regarding abdominal pain, vaginal bleeding and vaginal tissue loss containing her copper intra-uterine device (IUD). She used a contraceptive pill for heavy bleedings in combination with the IUD. Vaginal ultrasound showed no pregnancy. The gynaecologist diagnosed the tissue as a decidual cast.

Evaluation of photocrosslinked Lutrol hydrogel for tissue printing applications.

Application of hydrogels in tissue engineering and innovative strategies such as organ printing, which is based on layered 3D deposition of cell-laden hydrogels, requires design of novel hydrogel matrices. Hydrogel demands for 3D printing include: 1) preservation of the printed shape after the deposition; 2) maintaining cell viability and cell function and 3) easy handling of the printed construct. In this study we analyze the applicability of a novel, photosensitive hydrogel (Lutrol) for printing of 3D structured bone grafts. We benefit from the fast temperature-responsive gelation ability of thermosensitive Lutrol-F127, ensuring organized 3D extrusion, and the additional stability provided by covalent photocrosslinking allows handling of the printed scaffolds. We studied the cytotoxicity of the hydrogel and osteogenic differentiation of embedded osteogenic progenitor cells. After photopolymerization of the modified Lutrol hydrogel, cells remain viable for up to three weeks and retain the ability to differentiate. Encapsulation of cells does not compromise the mechanical properties of the formed gels and multilayered porous Lutrol structures were successfully printed.

Synthesis and characterization of hydroxyl-functionalized caprolactone copolymers and their effect on adhesion, proliferation, and differentiation of human mesenchymal stem cells.

The aim of this study was to develop new hydrophilic polyesters for tissue engineering applications. In our approach, poly(benzyloxymethyl glycolide-co-epsilon-caprolactone)s (pBHMG-CLs) were synthesized through melt copolymerization of epsilon-caprolactone (CL) and benzyl-protected hydroxymethyl glycolide (BHMG). Deprotection of the polymers yielded copolymers with pendant hydroxyl groups, poly(hydroxymethylglycolide-co-epsilon-caprolactone) (pHMG-CL). The synthesized polymers were characterized by GPC, NMR, and DSC techniques. The resulting copolymers consisting of up to 10% of HMG monomer were semicrystalline with a melting temperature above body temperature. Water contact angle measurements of polymeric films showed that increasing HMG content resulted in higher surface hydrophilicity, as evidenced from a decrease in receding contact angle from 68 degrees for PCL to 40 degrees for 10% HMG-CL. Human mesenchymal stem cells showed good adherence onto pHMG-CL films as compared to the more hydrophobic PCL surfaces. The cells survived and were able to differentiate toward osteogenic lineage on pHMG-CL surfaces. This study shows that the aforementioned hydrophilic polymers are attractive candidates for the design of scaffolds for tissue engineering applications.

Photopolymerized thermosensitive hydrogels: synthesis, degradation, and cytocompatibility.

In situ forming hydrogels based on thermosensitive polymers have attractive properties for tissue engineering. However, the physical interactions in these hydrogels are not strong enough to yield gels with sufficient stability for many of the proposed applications. In this study, additional covalent cross-links were introduced by photopolymerization to improve the mechanical properties and the stability of thermosensitive hydrogels. Methacrylate groups were coupled to the side chains of triblock copolymers (ABA) with thermosensitive poly( N-(2-hydroxypropyl) methacrylamide lactate) A blocks and a hydrophilic poly(ethylene glycol) B block. These polymers exhibit lower critical solution temperature (LCST) behavior in aqueous solution and the cloud point decreased with increasing amounts of methacrylate groups. These methacrylate groups were photopolymerized above the LCST to render covalent cross-links within the hydrophobic domains. The mechanical properties of photopolymerized hydrogels were substantially improved and their stability was prolonged significantly compared to nonphotopolymerized hydrogels. Whereas non-UV-cured gels disintegrated within 2 days at physiological pH and temperature, the photopolymerized gels degraded in 10 to 25 days depending on the degree of cross-linking. To assess biocompatibility, goat mesenchymal stem cells were seeded on the hydrogel surface or encapsulated within the gel and they remained viable as demonstrated by a LIVE/DEAD cell viability/cytotoxicity assay. Expression of alkaline phosphatase and production of collagen I demonstrated the functionality of the mesenchymal stem cells and their ability to differentiate upon encapsulation. Due to the improved mechanical properties, stability, and adequate cytocompatibility, the photopolymerized thermosensitive hydrogels can be regarded as highly potential materials for applications in tissue engineering.

Hydrogels as extracellular matrices for skeletal tissue engineering: state-of-the-art and novel application in organ printing.

Organ printing, a novel approach in tissue engineering, applies layered computer-driven deposition of cells and gels to create complex 3-dimensional cell-laden structures. It shows great promise in regenerative medicine, because it may help to solve the problem of limited donor grafts for tissue and organ repair. The technique enables anatomical cell arrangement using incorporation of cells and growth factors at predefined locations in the printed hydrogel scaffolds. This way, 3-dimensional biological structures, such as blood vessels, are already constructed. Organ printing is developing fast, and there are exciting new possibilities in this area. Hydrogels are highly hydrated polymer networks used as scaffolding materials in organ printing. These hydrogel matrices are natural or synthetic polymers that provide a supportive environment for cells to attach to and proliferate and differentiate in. Successful cell embedding requires hydrogels that are complemented with biomimetic and extracellular matrix components, to provide biological cues to elicit specific cellular responses and direct new tissue formation. This review surveys the use of hydrogels in organ printing and provides an evaluation of the recent advances in the development of hydrogels that are promising for use in skeletal regenerative medicine. Special emphasis is put on survival, proliferation and differentiation of skeletal connective tissue cells inside various hydrogel matrices.

Multifocal squamous cell carcinoma arising in a Favre-Racouchot lesion - report of two cases and review of the literature.

BACKGROUND: Favre-Racouchot syndrome (nodular cutaneous elastosis with cysts and comedones) is a cutaneous disease characterized by coexistence of cysts, comedones and elastotic nodules in actinically damaged skin, typically on the face. Ultraviolet radiation plays a significant role in the development of the disease. Unilateral lesions have been described. MAIN OBSERVATION: In this report we present two cases of squamous cell carcinoma arising in a unilateral Favre-Racouchot plaque. Both patients, fair-skinned, elderly, with impaired immune function developed large, deep invasive tumors with perineural extension. CONCLUSIONS: Squamous cell carcinomas of large size and prominent invasive growth developing in immunocompromised individuals carry poor prognosis with regard to recurrence rate and metastasis. Manifestations of malignancy as described in this report, indicate the importance of close follow-up of patients with Favre-Racouchot syndrome.

The osteoinductive potential of printable, cell-laden hydrogel-ceramic composites.

Hydrogels used as injectables or in organ printing often lack the appropriate stimuli to direct osteogenic differentiation of embedded multipotent stromal cells (MSCs), resulting in limited bone formation in these matrices. Addition of calcium phosphate (CaP) particles to the printing mixture is hypothesized to overcome this drawback. In this study we have investigated the effect of CaP particles on the osteoinductive potential of cell-laden hydrogel-CaP composite matrices. To this end, apatitic nanoparticles have been included in Matrigel constructs where after the viability of embedded progenitor cells was assessed in vitro. In addition, the osteoinductive potential of cell-laden Matrigel containing apatitic nanoparticles was investigated in vivo and compared with composites containing osteoinductive biphasic calcium phosphate (BCP) microparticles after subcutaneous implantation in immunodeficient mice. Histological and immunohistochemical analysis of the tissue response as well as in vivo bone formation revealed that apatitic nanoparticles were osteoinductive and induced osteoclast activation, but without bone formation. The BCP particles were more effective in inducing elaborate bone formation at the ectopic location.

Biofabrication of osteochondral tissue equivalents by printing topologically defined, cell-laden hydrogel scaffolds.

Osteochondral defects are prone to induce osteoarthritic degenerative changes. Many tissue-engineering approaches that aim to generate osteochondral implants suffer from poor tissue formation and compromised integration. This illustrates the need for further improvement of heterogeneous tissue constructs. Engineering of these structures is expected to profit from strategies addressing the complexity of tissue organization and the simultaneous use of multiple cell types. Moreover, this enables the investigation of the effects of three-dimensional (3D) organization and architecture on tissue function. In the present study, we characterize the use of a 3D fiber deposition (3DF) technique for the fabrication of cell-laden, heterogeneous hydrogel constructs for potential use as osteochondral grafts. Changing fiber spacing or angle of fiber deposition yielded scaffolds of varying porosity and elastic modulus. We encapsulated and printed fluorescently labeled human chondrocytes and osteogenic progenitors in alginate hydrogel yielding scaffolds of 1×2 cm with different parts for both cell types. Cell viability remained high throughout the printing process, and cells remained in their compartment of the printed scaffold for the whole culture period. Moreover, distinctive tissue formation was observed, both in vitro after 3 weeks and in vivo (6 weeks subcutaneously in immunodeficient mice), at different locations within one construct. These results demonstrate the possibility of manufacturing viable centimeter-scaled structured tissues by the 3DF technique, which could potentially be used for the repair of osteochondral defects.

Scaffold porosity and oxygenation of printed hydrogel constructs affect functionality of embedded osteogenic progenitors.

Insufficient supply of oxygen and nutrients throughout the graft is considered one of the principal limitations in development of large, tissue-engineered bone grafts. Organ or tissue printing by means of three-dimensional (3D) fiber deposition is a novel modality in regenerative medicine that combines pore formation and defined cell placement, and is used here for development of cell-laden hydrogel structures with reproducible internal architecture to sustain oxygen supply and to support adequate tissue development. In this study we tested the effect of porosity on multipotent stromal cells (MSCs) embedded in hydrogel constructs printed with a 3D fiber deposition (3DF) machine. For this, porous and solid alginate hydrogel scaffolds, with MSCs homogeneously dispersed throughout the construct, were printed and analyzed in vitro for the presence of hypoxia markers, metabolism, survival, and osteogenic differentiation. We demonstrated that porosity promotes oxygenation of MSCs in printed hydrogel scaffolds and supported the viability and osteogenic differentiation of embedded cells. Porous and solid printed constructs were subsequently implanted subcutaneously in immunodeficient mice to analyze tissue formation in relation to hypoxia responses of embedded cells. Implantation of printed grafts resulted in ingrowth of vascularized tissue and significantly enhanced oxygenation of embedded MSCs. In conclusion, the introduction of pores significantly enhances the conductive properties of printed hydrogel constructs and contributes to the functionality of embedded osteogenic progenitors.

Distinct tissue formation by heterogeneous printing of osteo- and endothelial progenitor cells.

The organ- or tissue-printing approach, based on layered deposition of cell-laden hydrogels, is a new technique in regenerative medicine suitable to investigate whether mimicking the anatomical organization of cells, matrix, and bioactive molecules is necessary for obtaining or improving functional engineered tissues. Currently, data on performance of multicellular printed constructs in vivo are limited. In this study we illustrate the ability of the system to print intricate porous constructs containing two different cell types--endothelial progenitors and multipotent stromal cells--and show that these grafts retain heterogeneous cell organization after subcutaneous implantation in immunodeficient mice. We demonstrate that cell differentiation leading to the expected tissue formation occurs at the site of the deposited progenitor cell type. While perfused blood vessels are formed in the endothelial progenitor cell-laden part of the constructs, bone formation is taking place in the multipotent stromal cell-laden part of the printed grafts.

Organ printing: the future of bone regeneration?

In engineered bone grafts, the combined actions of bone-forming cells, matrix and bioactive stimuli determine the eventual performance of the implant. The current notion is that well-built 3D constructs include the biological elements that recapitulate native bone tissue structure to achieve bone formation once implanted. The relatively new technology of organ/tissue printing now enables the accurate 3D organization of the components that are important for bone formation and also addresses issues, such as graft porosity and vascularization. Bone printing is seen as a great promise, because it combines rapid prototyping technology to produce a scaffold of the desired shape and internal structure with incorporation of multiple living cell types that can form the bone tissue once implanted.

The role of endothelial progenitor cells in prevascularized bone tissue engineering: development of heterogeneous constructs.

In vitro prevascularization of bone grafts with endothelial progenitor cells (EPCs) is a promising strategy to improveimplant survival. In this study we show bone formation in constructs that contain multipotent stromal cells (MSCs) and EPCs. Early and late EPCs from peripheral blood and bone marrow of adult goats were characterized for differentiation markers and functional responses. EPCs from peripheral blood are more proliferative than bone-marrow-derived EPCs, express higher numbers of endothelial markers for longer periods of time, and form more intricate networks. We demonstrate that EPCs derived from peripheral blood contribute to osteogenic differentiation by MSCs in vitro, and that MSCs support the proliferation of EPCs and stabilize the formed cellular networks. In vivo, EPCs from peripheral blood assemble into early blood vessel networks, which are more pronounced in the presence of MSCs. These results show that the EPCs isolated from peripheral blood are suitable for prevascularization strategies, and that coseeding of EPCs and MSCs is favorable for bone formation after 6 weeks.

The effect of photopolymerization on stem cells embedded in hydrogels.

Photopolymerizable hydrogels, formed by UV-exposure of photosensitive polymers in the presence of photoinitiators, are widely used materials in tissue engineering research employed for cellular entrapment and patterning. During photopolymerization, the entrapped cells are directly exposed to polymer and photoinitiator molecules. To develop strategies that prevent potential photoexposure-damage to osteoprogenitor cells, it is important to further characterize the effects of photopolymerization on the exposed cells. In this study we analyzed the viability, proliferation and osteogenic differentiation of multipotent stromal cell (MSC) monolayers after exposure to UV-light in the presence of Irgacure 2959, a frequently used photoinitiator in tissue engineering research. Cell cycle progression, apoptosis and osteogenic differentiation of encapsulated goat MSCs were studied in photopolymerized methacrylate-derivatized hyaluronic acid hydrogel and methacrylated hyperbranched polyglycerol gel. We demonstrate adverse effects of photopolymerization on viability, proliferation and reentry into the cell cycle of the exposed cells in monolayers, whereas the MSCs retain the ability to differentiate towards the osteogenic lineage. We further show that upon encapsulation in photopolymerizable hydrogels the viability of the embedded cells is unaffected by the photopolymerization conditions, while osteogenic differentiation depends on the type of hydrogel used.

In vivo matrix production by bone marrow stromal cells seeded on PLGA scaffolds for ligament tissue engineering.

Ligament tissue engineering based on cell-seeded biomechanically functional constructs is a commonly studied strategy toward native anterior cruciate ligament replacement. Little is known about the survival and differentiation of the seeded cells after the transplantation. We applied retroviral genetic marking to trace implanted cells and studied their differentiation by species-specific immunolabeling of the extracellular matrix produced. Goat bone marrow stromal cells were transduced with a MoMuLV-based vector encoding the DeltaLNGFR gene. Transduced cells were seeded onto poly(lactic-co-glycolic acid) (PLGA) fibers and implanted subcutaneously into nude mice and left for various periods up to 6 weeks. Immunohistochemistry for LNGFR expression showed survival of the seeded cells after transplantation for up to 6 weeks. Immunohistochemistry for collagen type I and III showed the production of fibrous tissue inside the scaffolds. Moreover, using a goat-specific anti-collagen type III, donor-derived matrix could be demonstrated. We conclude that bone marrow stromal cells survived in vivo and at least partially differentiated after implantation.

Three-dimensional fiber deposition of cell-laden, viable, patterned constructs for bone tissue printing.

Organ or tissue printing, a novel approach in tissue engineering, creates layered, cell-laden hydrogel scaffolds with a defined three-dimensional (3D) structure and organized cell placement. In applying the concept of tissue printing for the development of vascularized bone grafts, the primary focus lies on combining endothelial progenitors and bone marrow stromal cells (BMSCs). Here we characterize the applicability of 3D fiber deposition with a plotting device, Bioplotter, for the fabrication of spatially organized, cell-laden hydrogel constructs. The viability of printed BMSCs was studied in time, in several hydrogels, and extruded from different needle diameters. Our findings indicate that cells survive the extrusion and that their subsequent viability was not different from that of unprinted cells. The applied extrusion conditions did not affect cell survival, and BMSCs could subsequently differentiate along the osteoblast lineage. Furthermore, we were able to combine two distinct cell populations within a single scaffold by exchanging the printing syringe during deposition, indicating that this 3D fiber deposition system is suited for the development of bone grafts containing multiple cell types.