Design of Experiments Approach to Engineer Cell-Secreted Matrices for Directing Osteogenic Differentiation

The presentation of extracellular matrix (ECM) proteins provides an opportunity to instruct the phenotype and behavior of responsive cells. Decellularized cell-secreted matrix coatings (DM) represent a biomimetic culture surface that retains the complexity of the natural ECM. Microenvironmental culture conditions alter the composition of these matrices and ultimately the ability of DMs to direct cell fate. We employed a design of experiments (DOE) multivariable analysis approach to determine the effects and interactions of four variables (culture duration, cell seeding density, oxygen tension, and media supplementation) on the capacity of DMs to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs). DOE analysis revealed that matrices created with extended culture duration, ascorbate-2-phosphate supplementation, and in ambient oxygen tension exhibited significant correlations with enhanced hMSC differentiation. We validated the DOE model results using DMs predicted to have superior (DM1) or lesser (DM2) osteogenic potential for naïve hMSCs. Compared to cells on DM2, hMSCs cultured on DM1 expressed 2-fold higher osterix levels and deposited 3-fold more calcium over 3 weeks. Cells on DM1 coatings also exhibited greater proliferation and viability compared to DM2-coated substrates. This study demonstrates that DOE-based analysis is a powerful tool for optimizing engineered systems by identifying significant variables that have the greatest contribution to the target output.     

Computational Model for Migration of a Cell Cluster in Three-Dimensional Matrices

This paper presents a first forced-based dynamics computer model of a cell cluster moving collectively in a 3D environment mimicking the extracellular matrix. In general, collective cell migration is a relevant part of the mechanisms for tissue repair, morphogenesis, and cancer invasion. Particularly in cancer, invasion occurs through multicellular 3D strands as well as collective cell clusters. Because cancer is a slow process, these clusters have not been carefully observed. However, the prevalence of this mechanism of cell locomotion makes it a target for study. Due to the different molecular mechanisms involved in this movement and the complex relations among them, a computer model would be of great use. The model presented here takes into account ligand concentration, matrix metalloproteinase activity, and cluster geometry based on experimental findings and experimentally validated single cell computer models; thus incorporating implicitly different underlying molecular properties. The velocity profiles of the cell clusters were recorded and analyzed. In particular seven different profiles are observed based on different participation of ligands, proteinases, and mechanical forces involved. The model is successful in showing potential effects of altering single variables in a system of cells in motion. Special emphasis is made on future directions for improvement and the variables to be potentially modulated to simulate particular physiological conditions.     

Analysis of the Basic Characteristics of Osteogenic and Chondrogenic Cell Lines Important for Tissue Engineering Implants

Bulletin of Experimental Biology and Medicine - We isolated and characterized cultures of bone and cartilage tissue cells of laboratory minipigs. The size and morphological features of adherent...     

Conditional Long-Term Survival after Autologous Hematopoietic Cell Transplantation for Diffuse Large B Cell Lymphoma

Autologous hematopoietic cell transplantation (AHCT) is standard therapy for patients with chemosensitive, relapsed, diffuse large B cell lymphoma (DLBCL). We performed a retrospective cohort study to delineate subsequent (conditional) and relative survival in 371 adult patients with DLBCL who underwent AHCT between 2000 and 2014 and had survived for 1, 2, 3, or 5 years after transplant. The probability of overall survival at 10 years after AHCT was 62%, 71%, 77%, and 86%, respectively, for the 4 cohorts, whereas that of progression-free survival (PFS) was 55%, 65%, 72%, and 81%, respectively. The respective cumulative incidence of nonrelapse mortality (NRM) at 10 years after transplantation was 13%, 12%, 11%, and 8%, respectively. In multivariable analysis, older age was associated with greater mortality risk among all but 5-year survivors; relapse within the landmark time was associated with greater mortality risk in all groups. Older age and relapse within the landmark time were associated with worse PFS in all groups. Standardized mortality ratio (SMR) was significantly higher than an age-, gender-, and race-matched general population, with the magnitude of SMR decreasing as the landmark time increased (4.0 for 1-year, 3.0 for 2-year, 2.4 for 3-year, and 1.8 for 5-year survivors). Our study provides information on long-term survival and prognosis that will assist in counseling patients with DLBCL who have received AHCT. Survival improves with longer time in remission post-transplant, although patients continue to remain at risk for NRM, underscoring the need for continued vigilance and prevention of late complications.     

Membrane-Targeting Approaches for Enhanced Cancer Cell Destruction with Irreversible Electroporation

Irreversible electroporation (IRE) is a promising technology to treat local malignant cancer using short, high-voltage electric pulses. Unfortunately, in vivo studies show that IRE suffers from an inability to destroy large volumes of cancer tissue without introduction of cytotoxic agents and/or increasing the applied electrical dose to dangerous levels. This research will address this limitation by leveraging membrane-targeting mechanisms that increase lethal membrane permeabilization. Methods that directly modify membrane properties or change the pulse delivery timing are proposed that do not rely on cytotoxic agents. This work shows that significant enhancement (67–75% more cell destruction in vitro and >100% treatment volume increase in vivo) can be achieved using membrane-targeting approaches for IRE cancer destruction. The methods introduced are surfactants (i.e., DMSO) and pulse timing which are low cost, non-toxic, and easy to be incorporated into existing clinical use. Moreover, when needed, these methods can also be combined with electrochemotherapy to further enhance IRE treatment efficacy.     

Enhanced Thermal Conductivity of Thermoplastics by Lamellar Crystal Alignment of Polymer Matrices

Thermal conductivity (TC) of injection‐molded main‐chain smectic liquid‐crystalline polymers and the composites containing hexagonal boron nitride (h‐BN) particles is investigated. Shear flow during injection molding induces alignment of chain‐folding lamellar crystals of polymer matrices, in which polymer chains are aligned in the normal direction (ND) with respect to the molding surface, thus leading to a high TC (1.2 W m^?1 K^?1) in the ND. The composites exhibit a dramatic enhancement of TC in not only the ND but also the in‐plane direction. The enhanced TC is much higher than that of common thermoplastic composites at comparable loading levels. These results indicate that the polymer matrices serve as effective heat conductors between h‐BN particles.     

Osteogenic sarcoma and androgens

Endocrinologic examination of 320 male patients with primary osteogenic sarcoma reveals predominance of hyperandrogenemia characterized by decreased levels of sex hormonebinding globulin, elevated testosterone concentration, and increased free androgen index. The occurrence of androgen receptors was similar in the cytosolic fraction of osteogenic sarcoma (56%), benign tumors and tumor-like bone lesions (50%), while their content was higher in osteogenic sarcoma, implying an unfavorable prognosis of metastasizing. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122 No. 9, pp. 305–310, September, 1996     

Mononuclear Cell Telomere Attrition Is Associated with Overall Survival after Nonmyeloablative Allogeneic Hematopoietic Cell Transplantation for Hematologic Malignancies

After allogeneic hematopoietic cell transplantation (allo-HCT), transplanted cells rapidly undergo multiple rounds of division. This may cause extensive telomere attrition, which could potentially prohibit further cell division and lead to increased mortality. We therefore characterized the development in telomere length after nonmyeloablative allo-HCT in 240 consecutive patients transplanted because of hematologic malignancies and tested the hypothesis that extensive telomere attrition post-transplant is associated with low overall survival. Telomere length was measured using quantitative PCR in mononuclear cells obtained from donors and recipients pretransplant and in follow-up samples from recipients post-transplant. Telomere attrition at 9 to 15 months post-transplant was calculated as the difference between recipient telomere length at 9 to 15 months post-transplant and donor pretransplant telomere length, divided by donor pretransplant telomere length. Although allo-HCT led to shorter mean telomere length in recipients when compared with donors, recipients had longer mean telomere length 9 to 15 months post-transplant than they had pretransplant. When compared with donor telomeres, recipients with extensive telomere attrition at 9 to 15 months post-transplant had low overall survival (10-year survival from 9 to 15 months post-transplant and onward: 68% in the tertile with least telomere attrition, 57% in the middle tertile, and 39% in the tertile with most attrition; log-rank P?=?.01). Similarly, after adjusting for potential confounders, recipients with extensive telomere attrition had high all-cause mortality (multivariable adjusted hazard ratio, 1.84 per standard deviation of telomere attrition at 9 to 15 months post-transplant; 95% confidence interval, 1.25 to 2.72; P?=?.002) and high relapse-related mortality (subhazard ratio, 2.07; 95% confidence interval, 1.14 to 3.76; P?=?.02). Taken together, telomere attrition may be a clinically relevant marker for identifying patients at high risk of mortality.     

Favorable Overall Survival with Fully Myeloablative Allogeneic Stem Cell Transplantation for Follicular Lymphoma

Allogeneic stem cell transplantation (Allo-SCT) remains an option for patients with follicular lymphoma (FL). We performed a retrospective analysis to examine long-term disease control and treatment-related mortality (TRM) in a group of patients that underwent transplant for clinically high-risk disease. Thirty-seven patients with indolent FL (follicular small cleaved [FSC], follicular mixed [FM] or FL grades 1 or 2 by WHO criteria) underwent allo-SCT. Patients were in a chemosensitive remission at the time of SCT. The conditioning regimen was typically busulfan-cyclophosphamide (BuCy). Cyclophosphamide-total body irradiation (TBI) was used for unrelated donor SCT. The median age at the time of transplant was 45 years (range: 24-58). The median number of prior chemotherapy regimens was 3 (range: 1-6). Thirty-seven patients received BuCy conditioning and 2 patients underwent reduced intensity conditioning SCT. Seventy-two percent of patients had a matched sibling donor. With a median follow-up of 63.5 months in survivors, the 5-year overall survival is 79.1% (95% confidence interval 66.3%-94.4%). TRM was 15.4%, with an additional case of mortality from breast cancer. These results demonstrate that in selected younger patients, a fully myeloablative allo-SCT utilizing BuCy conditioning provides excellent OS and disease control with low TRM.     

A Comparative Study on Morphochemical Properties and Osteogenic Cell Differentiation within Bone Graft and Coral Graft Culture Systems

The objective of this study was to compare the morphological and chemical composition of bone graft (BG) and coral graft (CG) as well as their osteogenic differentiation potential using rabbit mesenchymal stem cells (rMSCs) in vitro. SEM analysis of BG and CG revealed that the pores in these grafts were interconnected, and their micro-CT confirmed pore sizes in the range of 107-315 µm and 103-514 µm with a total porosity of 92% and 94%, respectively. EDS analysis indicated that the level of calcium in CG was relatively higher than that in BG. FTIR of BG and CG confirmed the presence of functional groups corresponding to carbonyl, aromatic, alkyl, and alkane groups. XRD results revealed that the phase content of the inorganic layer comprised highly crystalline form of calcium carbonate and carbon. Atomic force microscopy analysis showed CG had better surface roughness compared to BG. In addition, significantly higher levels of osteogenic differentiation markers, namely, alkaline phosphatase (ALP), Osteocalcin (OC) levels, and Osteonectin and Runx2, Integrin gene expression were detected in the CG cultures, when compared with those in the BG cultures. In conclusion, our results demonstrate that the osteogenic differentiation of rMSCs is relatively superior in coral graft than in bone graft culture system.     

Relapse after Allogeneic Hematopoietic Cell Transplantation for Multiple Myeloma: Survival Outcomes and Factors Influencing Them

Many patients with multiple myeloma (MM) eventually relapse even after allogeneic hematopoietic cell transplantation (alloHCT) for curative intent. Over the past decade, outcomes for patients with MM have improved significantly with the availability of new therapies, including next-generation proteasome inhibitors, immunomodulatory agents, and, more recently, monoclonal antibodies. Although several published studies have evaluated the outcomes of alloHCT for MM, the data on survival outcomes in patients with MM experiencing disease relapse following alloHCT are limited. In addition, the predictors for postrelapse survival in these patients are not known. In this study, we examined the outcomes of a single-center cohort of 60 patients with MM who experienced relapse or progression after alloHCT. In addition, we evaluated the use of salvage regimens for treatment of relapsed MM and analyzed the predictors for improved postrelapse survival. After a median follow-up of 2.2 years from the time of relapse, the median duration of postrelapse survival was 1.8 years (95% confidence interval [CI], 1.2 to 5.0 years). Patients received a median of 3 lines of therapy (range, 0 to 10) for treatment of MM beyond the post-alloHCT relapse/progression. Multivariate analysis identified cytogenetic risk (standard risk versus high risk; hazard ratio [HR], .34; P = .01), time to relapse after alloHCT (>12 months versus ≤12 months: HR, .41; P = .04), and occurrence of acute graft-versus-host disease (GVHD) before relapse (GVHD versus no GVHD: HR, 2.89; P = .01) significantly affected postrelapse survival. These data illustrate that long-term myeloma control and survival is attainable in those relapsing/progressing after alloHCT and suggest that the synergism between novel therapies and the allogeneic immune platform is the key to improved survival in this high-risk patient population.     

通过抑制自噬作用增强声动力学疗法诱导的肿瘤细胞凋亡研究

探讨声动力疗法诱导S180细胞凋亡过程中自噬现象的发生及其在细胞凋亡中的作用。用频率为1.1 MHz、功率为3 W/cm²的超声,结合1 μg/mL原卟啉Ⅸ作用于S180细胞,MTT检测细胞存活率;DAPI染色检测凋亡细胞核形态变化;Rhodamine 123 染色检测线粒体膜电位;Western blotting检测自噬标记分子LC3由Ⅰ型向Ⅱ型的转化;吖啶橙（AO）活细胞染色观察自噬小体。声动力处理后,S180细胞明显受损,MTT显示存活率为59.3%并且出现了典型的凋亡形态学特征,线粒体膜电位下降至MFI=971.28,LC3-II表达明显升高;利用自噬和凋亡的抑制剂实验结果提示自噬体的形成（1 h）早于细胞凋亡的发生 (8 h),自噬抑制剂3-MA和Ba A1增强了SDT对线粒体膜电位的破坏,DAPI染色进一步证实了自噬抑制剂有利于促进SDT诱导的S180细胞凋亡。提示自噬参与了SDT诱导的S180细胞死亡,自噬特异性药物抑制剂增强了SDT诱导的细胞凋亡。