Outcome of Scaphoid Nonunion Using Open Reduction and Internal Fixation With Iliac Crest Bone Graft (Fisk-Fernandez Technique).

Introduction The scaphoid is the most common carpal bone to be fractured and has a high propensity for nonunion. Restoration of scaphoid length mitigates the chances of long-term complications. The aim of this study was to assess the functional outcome of the Fisk-Fernandez technique for the treatment of scaphoid nonunion by using open reduction and internal fixation with trapezoidal iliac crest bone graft.  Materials and methods Fisk-Fernandez technique was used to manage scaphoid nonunion in 31 patients at a tertiary care hospital with follow-up at six weeks, 12 weeks, and 24 weeks. An objective assessment of the outcome was done using a comparison of the pre- and postoperative scaphoid score, QuickDASH, and visual analog score. Discussion The scaphoid is one of the most common carpal bones to get fractured. Anatomical factors, late presentation, and delay in diagnosis render it to usually land in nonunion. A comparison of the preoperative scaphoid, QuickDASH, and VAS scores with six-week, 12-week, and 24-week postoperative scores was made and was found to be statistically significant (p<0.001). Ninety-three percent of patients subjectively reported satisfaction after treatment. Though revascularization was not assessed, the bony union was observed in all the patients. Conclusion The operative technique proposed by Fisk-Fernandez is effective in correcting deformity of the scaphoid as well as providing satisfactory functional outcomes in patients with scaphoid nonunion.

Fluorescence-based actin turnover dynamics of stem cells as a profiling method for stem cell functional evolution, heterogeneity and phenotypic lineage parsing.

Stem cells are widely explored in regenerative medicine as a source to produce diverse cell types. Despite the wide usage of stem cells like mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs), there is a lack of robust methods to rapidly discern the phenotypic and functional heterogeneity of stem cells. The organization of actin cytoskeleton has been previously used to discern divergent stem cell differentiation pathways. In this paper, we highlight the versatility of a cell profiling method for actin turnover dynamics. Actin filaments in live stem cells are labeled using SiR-actin, a cell permeable fluorogenic probe, to determine the endogenous actin turnover. Live MSC imaging after days of induction successfully demonstrated lineage specific change in actin turnover. Next, we highlighted the differences in the cellular heterogeneity of actin dynamics during adipogenic or osteogenic MSC differentiation. Next, we applied the method to differentiating iPSCs in culture, and detected a progressive slowdown in actin turnover during differentiation upon stimulation with neural or cardiac media. Finally, as a proof of concept, the actin dynamic profiling was used to isolate MSCs via flow cytometry prior to sorting into three distinct sub-populations with low, intermediate or high actin dynamics. A greater fraction of MSCs with more rapid actin dynamics demonstrated increased inclination for adipogenesis, whereas, slower actin dynamics correlated with increased osteogenesis. Together, these results show that actin turnover can serve as a versatile biomarker to not only track cellular phenotypic heterogeneity but also harvest live cells with potential for differential phenotypic fates.

Fluorescence Imaging of Actin Turnover Parses Early Stem Cell Lineage Divergence and Senescence.

This study describes a new approach to discern early divergence in stem cell lineage progression via temporal dynamics of the cytoskeletal protein, F-actin. The approach involves real-time labeling of human mesenchymal stem cells (MSCs) and longitudinal tracking of the turnover dynamics of a fluorogenic F-actin specific probe, SiR-actin (SA). Cells cultured in media with distinct lineage factors and labeled with SA showed lineage specific reduction in the actin turnover shortly after adipogenic (few minutes) and chondrogenic (3-4 hours) commitment in contrast to osteogenic and basal cultured conditions. Next, composite staining of SA along with the competing F-actin specific fluorescent conjugate, phalloidin, and high-content image analysis of the complementary labels showed clear phenotypic parsing of the sub-populations as early as 1-hour post-induction across all three lineages. Lastly, the potential of SA-based actin turnover analysis to distinguish cellular aging was explored. In-vitro aged cells were found to have reduced actin turnover within 1-hour of simultaneous analysis in comparison to cells of earlier passage. In summary, SiR-actin fluorescent reporter imaging offers a new platform to sensitively monitor emergent lineage phenotypes during differentiation and aging and resolve some of the earliest evident differences in actin turnover dynamics.

Substrate micropatterns produced by polymer demixing regulate focal adhesions, actin anisotropy, and lineage differentiation of stem cells.

Stem cells are adherent cells whose multipotency and differentiation can be regulated by numerous microenvironmental signals including soluble growth factors and surface topography. This study describes a simple method for creating distinct micropatterns via microphase separation resulting from polymer demixing of poly(desaminotyrosyl-tyrosine carbonate) (PDTEC) and polystyrene (PS). Substrates with co-continuous (ribbons) or discontinuous (islands and pits) PDTEC regions were obtained by varying the ratio of PDTEC and sacrificial PS. Human mesenchymal stem cells (MSCs) cultured on co-continuous PDTEC substrates for 3 days in bipotential adipogenic/osteogenic (AD/OS) induction medium showed no change in cell morphology but exhibited increased anisotropic cytoskeletal organization and larger focal adhesions when compared to MSCs cultured on discontinuous micropatterns. After 14 days in bipotential AD/OS induction medium, MSCs cultured on co-continuous micropatterns exhibited increased expression of osteogenic markers, whereas MSCs on discontinuous PDTEC substrates showed a low expression of adipogenic and osteogenic differentiation markers. Substrates with graded micropatterns were able to reproduce the influence of local underlying topography on MSC differentiation, thus demonstrating their potential for high throughput analysis. This work presents polymer demixing as a simple, non-lithographic technique to produce a wide range of micropatterns on surfaces with complex geometries to influence cellular and tissue regenerative responses. STATEMENT OF SIGNIFICANCE: A better understanding of how engineered microenvironments influence stem cell differentiation is integral to increasing the use of stem cells and materials in a wide range of tissue engineering applications. In this study, we show the range of topography obtained by polymer demixing is sufficient for investigating how surface topography affects stem cell morphology and differentiation. Our findings show that co-continuous topographies favor early (3-day) cytoskeletal anisotropy and focal adhesion maturation as well as long-term (14-day) expression of osteogenic differentiation markers. Taken together, this study presents a simple approach to pattern topographies that induce divergent responses in stem cell morphology and differentiation.

The overwhelming use of rat models in nerve regeneration research may compromise designs of nerve guidance conduits for humans.

Rats are not the best model for the evolving complexities we face in designing nerve repair strategies today. The development of effective nerve guidance conduits for nerve regeneration is severely limited by the rat sciatic nerve model as the almost exclusive research model in academia. An immense effort is underway to develop an alternative to autologous nerve grafts for the repair of nerve defects, aiming particularly at larger gap repairs of 5-30 cm or more. This must involve combinations of ever more complex components, which in the vast majority of cases begin their testing in the rat model. Three major problems are at play: (1) The majority of nerve regeneration data is now being generated in the rat, which is likely to skew treatment outcomes and lead to inappropriate evaluation of risks and benefits. (2) The rat is a particularly poor model for the repair of human critical gap defects due to both its small size and its species-specific neurobiological regenerative profile. (3) Translation from rat to human has proven unreliable for nerve regeneration, as for many other applications. We explore each of these facets and their implications, in order to highlight the need for appropriate awareness in animal model selection when translating nerve regeneration modalities of ever-increasing complexity-from relatively simple devices to drug-device-biologic combinations.

The half-life of the HSV-1 1.5-kb LAT intron is similar to the half-life of the 2.0-kb LAT intron.

Herpes simplex virus type 1 establishes a latent infection in the sensory neurons of the peripheral nervous system of humans. Although about 80 genes are expressed during the lytic cycle of the virus infection, essentially only one gene is expressed during the latent cycle. This gene is known as the latency-associated transcript (LAT), and it appears to play a role in the latency cycle through an anti-apoptotic function in the 5' end of the gene and miRNA encoded along the length of the transcript which downregulate some of the viral immediate-early gene products. The LAT gene is about 8.3 kb long and consists of two exons separated by an unusual intron. The intron between the exons consists of two nested introns. This arrangement of introns has been called a twintron. Furthermore, the larger (2 kb) intron has been shown to be very stable. In this study, we measure the stability of the shorter 1.5-kb nested intron and find its half-life is similar to the longer intron. This was achieved by deleting the 0.5-kb overlapping intron from a plasmid construct designed to express the LAT transcript from a tet-inducible promoter and measuring the half-life of the 1.5-kb intron in tissue culture cells. This finding supports the hypothesis that it is the common branch-point region of these nested introns that is responsible for their stability.