Apoptosis in bone for tissue engineering

Bone loss due to congenital defects, trauma, improper fracture fixation, metabolic disturbances, infections, or after tumor resection represents a major clinical problem in head and neck surgery. To address these issues, different types of scaffolds, growth factors and cell sources -- alone or in various combinations -- have been applied for development of bioartificial bone tissues. Although these applications have received increasing interest, use of autologous bone grafts is still considered as the gold standard for tissue repair. Despite progress in some areas of tissue regeneration, significant translation into clinical practice has not been achieved. Reasons for this impass include rejection of engineered tissue implants by the immune system, limited blood supply, or morbidity of the donor site. During the process of bone regeneration, approximately 50-70% of osteoblasts undergo apoptosis. Apoptosis is a naturally occurring cell death pathway induced in a variety of cell types and is associated with caspase activation or caspase mediation. It is recognized as an important component of embryogenesis and tissue morphogenesis and, in adult skeletons, it contributes substantially to physiological bone turnover, repair, and regeneration. Intracellular mechanisms are orchestrated by a variety of proteins, the interplay of which seems to vary, depending on the differentiation state of the cell or the current status of the tissue. Closing gaps in current knowledge of the apoptosis of bone and understanding the mechanisms of cell death in tissue engineered bone will improve results in the translation from bench to bedsite. This review aims to provide a broad overview of the current general concepts in apoptosis with a special focus on its regulation in osteoblasts and its significance for bone tissue engineering.     

Genetic variation in arsenic (+3 oxidation state) methyltransferase (AS3MT), arsenic metabolism and risk of basal cell carcinoma in a European population

Exposure to inorganic arsenic increases the risk of basal cell carcinoma (BCC). Arsenic metabolism is a susceptibility factor for arsenic toxicity, and specific haplotypes in arsenic (+3 oxidation state) methyltransferase (AS3MT) have been associated with increased urinary fractions of the most toxic arsenic metabolite, methylarsonic acid (MMA). The aim of this study is to elucidate the association of AS3MT haplotypes with arsenic metabolism and the risk of BCC. Four AS3MT polymorphisms were genotyped in BCC cases (N = 529) and controls (N = 533) from Eastern Europe with low to moderate arsenic exposure (lifetime average drinking water concentration: 1.3 µg/L, range 0.01–167 µg/L). Urinary metabolites [inorganic arsenic (iAs), MMA, dimethylarsinic acid (DMA)] were analyzed by HPLC‐ICPMS. Five AS3MT haplotypes (based on rs3740400 A/C, rs3740393 G/C, rs11191439 T/C and rs1046778 T/C) had frequencies >5%. Individuals with the CCTC haplotype had lower %iAs (P = 0.032) and %MMA (P = 0.020) in urine, and higher %DMA (P = 0.033); individuals with the CGCT haplotype had higher %MMA (P < 0.001) and lower %DMA (P < 0.001). All haplotypes showed increased risk of BCC with increasing arsenic exposure through drinking water (ORs 1.1–1.4, P values from <0.001 to 0.082), except for the CCTC haplotype (OR 1.0, CI 0.9–1.2, P value 0.85). The results suggest that carriage of AS3MT haplotypes associated with less‐efficient arsenic methylation, or lack of AS3MT haplotypes associated with a more‐efficient arsenic methylation, results in higher risk of arsenic‐related BCC. The fact that AS3MT haplotype status modified arsenic metabolism, and in turn the arsenic‐related BCC risk, supports a causal relationship between low‐level arsenic exposure and BCC. Environ. Mol. Mutagen. 56:60–69, 2015. © 2014 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society     

Integrin expression in stem cells from bone marrow and adipose tissue during chondrogenic differentiation

The use of adult mesenchymal stem cells (MSC) in cartilage tissue engineering offers new perspectives in the generation of transplants for reconstructive surgery. The extracelular matrix (ECM) plays a key role in modulating the function and phenotype of the embedded cells and contains the integrins as adhesion receptors mediating cell-cell and cell-matrix interactions. In our study, characteristic changes in integrin expression during the course of chondrogenic differentiation of MSC from bone marrow and adipose tissue were compared. MSC were isolated from bone marrow biopsies and adipose tissue. During cell culture, chondrogenic differentiation was performed. The expression of integrins and their signaling components were analysed with microarray and immunohistochemistry in freshly isolated MSC and after chondrogenic differentiation. The fibronectin receptor (integrin alpha5beta1) was expressed by undifferentiated MSC, and expression rose during chondrogenic differentiation in both types of MSC. The components of the vitronectin/osteopontin receptors (alphavbeta5) were not expressed by freshly isolated MSC, and expression rose with ongoing differentiation. Receptors for the collagens (alpha1beta1, alpha2beta1, alpha3beta1) were weakly expressed by undifferentiated MSC and were activated during differentiation. Intracellular signaling components integrin-linked kinase (ILK) and CD47 showed increased expression with ongoing differentiation. For all integrins, no significant differences were be found in the 2 types of MSC. Integrin-mediated signaling appeared to play an important role in the generation and maintenance of the chondrocytic phenotype during chondrogenic differentiation. Particularly, the receptors for fibronectin, vitronectin, osteopontin and the collagens may be involved in the generation of the ECM. Intracellularly, their signals might be transduced by ILK and CD47. To fully harness the potential of these cells, future studies should be directed to ascertain their cellular and molecular characteristics for optimal identification, isolation, and expansion.     

Characterization of human myoblast cultures for tissue engineering

Skeletal muscle tissue engineering, a promising specialty, aims at the reconstruction of skeletal muscle loss. In vitro tissue engineering attempts to achieve this goal by creating differentiated, functional muscle tissue through a process in which stem cells are extracted from the patient, e.g. by muscle biopsies, expanded and differentiated in a controlled environment, and subsequently re-implanted. A prerequisite for this undertaking is the ability to cultivate and differentiate human skeletal muscle cell cultures. Evidently, optimal culture conditions must be investigated for later clinical utilization. We therefore analysed the proliferation of human cells in different environments and evaluated the differentiation potential of different culture media. It was shown that human myoblasts have a higher rate of proliferation in the alamarBlue assay when cultured on gelatin-coated culture flasks rather than polystyrene-coated flasks. We also demonstrated that myoblasts treated with a culture medium with a high concentration of growth factors [growth medium (GM)] showed a higher proliferation compared to cultures treated with a culture medium with lower amounts of growth factors [differentiation medium (DM)]. Differentiation of human myoblast cell cultures treated with GM and DM was analysed until day 16 and myogenesis was verified by expression of MyoD, myogenin, alpha-sarcomeric actin and myosin heavy chain by semi-quantitative RT-PCR. Immunohistochemical staining for desmin, Myf-5 and alpha-sarcomeric actin was performed to verify the myogenic phenotype of extracted satellite cells and to prove the maturation of cells. Cultures treated with DM showed positive staining for alpha-sarcomeric actin. Notably, markers of differentiation were also detected in cultures treated with GM, but there was no formation of myotubes. In the enzymatic assay of creatine phosphokinase, cultures treated with DM showed a higher activity, evidencing a higher degree of differentiation. In this study, we obtained detailed information regarding the cultivation and differentiation of human myoblast cultures in different environments. By exploring optimal culture conditions for skeletal muscle tissue engineering, we acquired culture data for comparison with other sources of stem cells in order to find the most applicable stem cell for focussed clinical usage.