Osteopontin Rejuvenates Senescent Adipose-Derived Stem Cells and Restores their Bone Tissue Regenerative Function.

The regenerative function of stem cells is compromised when the proportion of senescent stem cells increases with ageing advance. Therefore, combating stem cell senescence is of great importance for stem cell-based tissue engineering in the elderly, but remains largely unexplored. Osteopontin (OPN), a glycosylated phosphoprotein, is one of the key extracellular matrix molecules in bone tissue. OPN activates various signalling pathways and modulates cellular activities, including cell senescence. However, the role of OPN in stem cell senescence remains largely unknown. This study aims to investigate if OPN modulates cell senescence and bone regenerative function in human adipose-derived mesenchymal stem cells (ASCs), and to determine the underlying mechanisms. We first developed a senescent ASC model using serial passaging until passage 10 (P10), in which senescent cells were characterised by reduced proliferation and osteogenic differentiation capacity compared to P4 ASCs. The conditioned medium from P10 ASCs exhibited a diminished trophic effect on human osteoblasts (HOBs), compared to that from P4 ASCs. P10 ASCs on OPN-coated surface showed rejuvenated phenotype and enhanced osteogenic differentiation. The conditioned medium from P10 ASCs on OPN-coating improved trophic effects on HOBs. OPN regulated the morphology of senescent ASCs, transforming them from a more rounded and flattened cell shape to an elongated shape with a smaller area. These findings demonstrated the effects of OPN in restoring senescent ASCs functions, possibly through a mechanism that involves the modulation of cell morphology, indicating that OPN might hold a great potential for rejuvenating senescent stem cells and could potentially open a new venue for regenerating bone tissue in age-related diseases.

Model-based Bayesian inference under computer assisted balance-improving designs.

To improve covariate balance over a complete randomization, a number of methods have been proposed recently to utilize modern computational capabilities to find allocations with balance in observed covariates. Asymptotic inference on treatment effects based on these designs is more complicated than that under complete randomization, and this is why Fisher randomization tests often are suggested. This article suggests model-based Bayesian inference as a general method of inference in these designs, which can deal with complications such as arbitrary covariate balancing criteria and complex estimands. As an illustration, we focus on the case when the outcome is linearly related to the covariates and the estimand of interest is the Sample Average Treatment Effect (SATE). We use a large Monte Carlo simulation to compare the finite sample performance of the model-based Bayesian inference with that of two previous methods which are valid for asymptotic inference of SATE under Mahalanobis distance based rerandomization. We find that for experiments with small to moderate sample sizes, Bayesian inference is to be preferred to the previous methods. As a byproduct, we also find that regression adjustment together with small sample adjusted estimators of standard errors perform better than the previous methods.

Dual-enzymatically cross-linked gelatin hydrogel enhances neural differentiation of human umbilical cord mesenchymal stem cells and functional recovery in experimental murine spinal cord injury.

Recently, an advanced stem cell and tissue engineering approach has been recognized as an emerging and fascinating strategy to promote neural repair in spinal cord injury (SCI). Hydrogels can be properly engineered to encapsulate cells, enhance cell viability and neural differentiation, and provide the advantage of flexible adaptation to irregular defects. In this study, a dual-enzymatically cross-linked gelatin hydrogel with hydrogen horseradish peroxidase (HRP) and galactose oxidase (GalOx) was proposed to combine human umbilical cord mesenchymal stem cells (hUC-MSCs) for facilitating nerve regeneration post-SCI. In vitro, hUC-MSCs in this 3D gelatin hydrogel displayed good viability, proliferation, and neuronal differentiation. To further evaluate the neural regeneration effect of hUC-MSCs loaded into gelatin hydrogels in vivo, a clinically-relevant and force-controlled contusion model of mouse spinal cords was established. We found that implantation of a hydrogel loaded with hUC-MSCs significantly promoted the motor function recovery evaluated by Basso Mouse Scale (BMS) and footprint tests. Further histological analysis showed that the hydrogel and hUC-MSC combined transplantation dramatically decreased inflammation, inhibited apoptosis and promoted neurogenesis. Overall, implantation of this dual-enzymatically cross-linked and MSC-laden 3D gelatin hydrogel is a promising therapeutic strategy for SCI treatment.

Control the fate of human umbilical cord mesenchymal stem cells with dual-enzymatically cross-linked gelatin hydrogels for potential applications in nerve regeneration.

Stem-cell-based therapy is a promising strategy to treat challenging neurological diseases, while its application is hindered primarily by the low viability and uncontrolled differentiation of stem cell. Hydrogel can be properly engineered to share similar characteristics with the target tissue, thus promoting cell viability and directing cell differentiation. In this study, we proposed a new dual-enzymatically cross-linked and injectable gelatin hydrogel for regulating survival, proliferation, and differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs) in a three-dimensional matrix. This injectable gelatin hydrogel was formed by oxidative coupling of gelatin-hydroxyphenyl acid conjugates catalyzed by hydrogen horseradish peroxidase (HRP) and choline oxidase (ChOx). Modulus and H2 O2 release can be well controlled by ChOx activity. Results from calcein-AM/PI staining and Ki67 immunofluorescence tests demonstrated that the survival and proliferation behavior of hUC-MSCs were highly enhanced in HRP1U ChOx0.25U hydrogel with lower modulus and less H2 O2 release compared with other groups. Attractively, the expression of neuron-specific markers ß-III tubulin, neurofilament light chain (NFL), and synapsin-1 was significantly increased in HRP1U ChOx0.25U hydrogel as well. Additionally, in vitro hemolysis test and in vivo HE staining data highlighted the good biocompatibility. Undoubtedly, this injectable gelatin hydrogel's ability to control hUC-MSCs' fate holds enormous potentials in nervous disorders' therapy and nerve regeneration.

A chitosan-based thermosensitive scaffold loaded with bone marrow-derived mesenchymal stem cells promotes motor function recovery in spinal cord injured mice.

Spinal cord injury is a devastating trauma with high mortality and disability, for which there is no effective treatment. Stem cell-based tissue engineering has been reported to promote functional neural recovery. At present, building a neural scaffold with excellent biocompatibility for cells and tissues is still challenging. In this study, a new thermosensitive composite hydrogel based on chitosan, hydroxyethyl cellulose, collagen and ß-phosphoglycerate (CS-HEC-Col/GP hydrogel) is developed to encapsulate murine bone marrow-derived mesenchymal stem cells (BMSCs) to improve therapeutic efficacy in spinal cord injured mice. This composite hydrogel possesses a good cytocompatibility to mouse BMSCs by live/dead staining, minimized inflammatory reaction in vivo by hematoxylin and eosin staining and suitable rheological behavior similar to neural tissue, ranging from 100 to 1000 Pa. Furthermore, the data from animal experiments indicated that BMSC-loaded CS-HEC-Col/GP hydrogel could enhance the survival or proliferation of endogenous nerve cells, probably by secreting neurotrophic factors and inhibiting apoptosis, and thereby promote the recovery of motor function in the hindlimbs of a murine spinal cord injury model.

Dual-enzymatically crosslinked hyaluronic acid hydrogel as a long-time 3D stem cell culture system.

Stem cell-based tissue engineering shows enormous potential for regenerative medicine. Three-dimensional (3D) stem cell culture is the most basic aspect of tissue engineering. However, achievement of a perfect scaffold for highly efficient 3D cell culture is currently still limited. Herein, a new hyaluronic acid hydrogel dual-enzymatically crosslinked by horseradish peroxidase and choline oxidase is developed as a 3D stem cell culture system. This hydrogel possesses superior stability over two months, controllable biodegradability with hyaluronidases, a high swelling ratio exceeding 6000%, and excellent cytocompatibility in vitro and biocompatibility in vivo. More importantly, a long-time and highly cellular activity 3D culture of bone marrow-derived mesenchymal stem cells was achieved in vitro over 20 days. All these encouraging results highlight the great potential of this new hydrogel for 3D culture and tissue engineering.

Dual-enzymatically crosslinked and injectable hyaluronic acid hydrogels for potential application in tissue engineering.

Recently, in situ formed injectable hydrogels have shown great potential in biomedical applications as therapeutic implants or carriers in tissue repair and regeneration. They can seal or fill the damaged tissue to function as cell/drug delivery vehicle perfectly through a minimally invasive surgical procedure. In this study, hyaluronic acid (HA) is functionalized with tyramine to produce an injectable hydrogel dual-enzymatically crosslinked by horseradish peroxidase (HRP) and galactose oxidase (GalOX). This new tyramine-modified HA (HT) hydrogel exhibited good injectability, favorable cytocompatibility to mice bone marrow mesenchymal stem cells (BMSCs), and low inflammatory response verified by cytotoxicity assay in vitro and an in situ subcutaneous injection study in vivo. In addition, the gelation time, swelling behavior, and degradation rate of the HT hydrogel could be adjusted through varying the concentrations of HT and GalOX in a certain range. These encouraging results suggest that such biocompatible HT hydrogels might have potential application in three-dimensional stem cell culture and tissue engineering.

Subgroup balancing propensity score.

This paper concerns estimation of subgroup treatment effects with observational data. Existing propensity score methods are mostly developed for estimating overall treatment effect. Although the true propensity scores balance covariates in any subpopulations, the estimated propensity scores may result in severe imbalance in subgroup samples. Indeed, subgroup analysis amplifies a bias-variance tradeoff, whereby increasing complexity of the propensity score model may help to achieve covariate balance within subgroups, but it also increases variance. We propose a new method, the subgroup balancing propensity score, to ensure good subgroup balance as well as to control the variance inflation. For each subgroup, the subgroup balancing propensity score chooses to use either the overall sample or the subgroup (sub)sample to estimate the propensity scores for the units within that subgroup, in order to optimize a criterion accounting for a set of covariate-balancing moment conditions for both the overall sample and the subgroup samples. We develop two versions of subgroup balancing propensity score corresponding to matching and weighting, respectively. We devise a stochastic search algorithm to estimate the subgroup balancing propensity score when the number of subgroups is large. We demonstrate through simulations that the subgroup balancing propensity score improves the performance of propensity score methods in estimating subgroup treatment effects. We apply the subgroup balancing propensity score method to the Italy Survey of Household Income and Wealth (SHIW) to estimate the causal effects of having debit card on household consumption for different income groups.

New BMSC-Laden Gelatin Hydrogel Formed in Situ by Dual-Enzymatic Cross-Linking Accelerates Dermal Wound Healing.

In situ forming hydrogel shows enormous potential as a therapeutic implant or carrier in tissue repair and regeneration. It can perfectly seal or fill the defective tissue, consequently functioning as a cell/drug delivery vehicle. In this contribution, a new gelatin hydrogel with dual-enzymatic cross-linking of horseradish peroxidase (HRP) and galactose oxidase (GalOx) was developed, and the therapeutic effect of this hydrogel encapsulated with bone mesenchymal stem cells (BMSC) in dermal wound healing was investigated. This hydrogel possesses a quick gelation process within 5 min, a high water content, and a uniform three-dimensional (3D) porous network. The 3D cell culture study indicated that gelatin hydrogel matrix of HRP(5U):GalOx(1U) or HRP(2U):GalOx(1U) could provide a friendly 3D microenvironment for supporting the survival, proliferation, and spread of mouse bone mesenchymal stem cells (BMSC) with negligible cytotoxicity. Hematoxylin and eosin staining test suggested that this hydrogel has superior histocompatibility and minimized immune response in vivo. Furthermore, wound-healing studies on a C57 mouse model of excised wound demonstrated that BMSC-laden gelatin hydrogel could significantly accelerate the wound closure as compared to other groups. These data suggest that this dual-enzymatically cross-linked gelatin hydrogel loaded with BMSC has a great potential in wound healing and other tissue-regeneration applications.

A dual-enzymatically cross-linked injectable gelatin hydrogel loaded with BMSC improves neurological function recovery of traumatic brain injury in rats.

Injectable hydrogels can perfectly fill defective areas and thereby show promise as therapeutic implants or cell/drug delivery vehicles in traumatic brain injury (TBI) or other tissue repair. In this study, an injectable gelatin hydrogel in situ dual-enzymatically cross-linked by glucose oxidase (GOX) and horseradish peroxidase (HRP) was synthesized and optimized, and the therapeutic effect of this hydrogel encapsulated with bone mesenchymal stem cells (BMSC) for TBI rats was investigated. By changing the GOX content, we found that this hydrogel cross-linked by 0.1 U mL-1 GOX possesses better cytocompatibility than that with a higher GOX content, and minimized immune response in vivo. More importantly, this hydrogel loaded with BMSC could significantly accelerate the healing process of a damaged region, facilitate the survival and proliferation of endogenous nerve cells probably by nutrition supply and apoptosis suppression, and consequently promote the neurological function recovery of the TBI rat model. All these findings highlight that this injectable and BMSC-laden gelatin hydrogel has enormous potential for TBI therapy and other tissue regeneration strategies.

[The detection of microsatellite instability by ion-pair reversed-phase high performance liquid chromatography].

OBJECTIVE: To set up a sensitive and stable technique which has high throughout to detect the instability of microsatellite DNA. METHODS: Genomic DNA extracted from the cancer tissues and their normal tissues were subjected to microsatellite instability(MSI) analysis on five of DNA markers in 115 sporadic colorectal cancers by means of PCR and ion-pair reversed-phase high performance liquid chromatography. Genomic DNA extracted from lymphocytes in blood of 20 normal persons were analysed and used as the standard control. RESULTS: Seventeen (14.8%) MSI-H and 23(20.0%) MSI-L were found in 115 sporadic colorectal cancers. The rates of MSI in the young patients and old patients were much higher than that in the middle-age patients (P<0.05). And the rate of MSI in low differentiation group was also much higher than that in high or middle differentiation groups (P<0.05). CONCLUSION: The method the authors developed is a sensitive and accurate technique to detect MSI and has a high throughput.

[Large deletion in mismatch repair genes uncovered by quantitative multiplex PCR-high performance liquid chromatography system].

OBJECTIVE: Establishing a new method on the basis of multiplex PCR-high performance liquid chromatography (HPLC) for screening a large deletion in mismatch repair genes. METHODS: Thirty-five pairs of primers were used to amplify all 16 exons of MSH2 and all 19 exons of MLH1 gene in 8 multiplex PCR. The products of multiplex PCR were analysed for the large deletion with Double Strand DNA Analysis System of HPLC. Firstly, validation of the method was tested on positive and negative controls in blind analysis. Secondly, 14 blood cell DNA samples from hereditary nonpolyposis colorectal cancer (HNPCC) patients and 13 colorectal cancer (CRC) tissues DNA samples from sporadic CRC patients were checked with the new developed method. RESULTS: (1) the genomic deletions in all 4 of positive controls were identically uncovered with the new method; (2) a novel germline and a novel somatic large deletions were unveiled in 1/14 HNPCC patients and in 1/13 CRC tissues. CONCLUSION: The method developed on multiplex PCR-HPLC is reliable for uncovering large genomic deletion in mismatch repair genes, and can be taken as a valuable addition to mutation screening system.

[Mutation analysis on MSH2 and MLH1 genes in patients of colorectal cancer at early age].

OBJECTIVE: To investigate the etiological role of function and construction alteration in mismatch repair genes MSH2 and MLH1 in the patients onset of colorectal cancers (CRC) at early ages. METHODS: The genomic DNA was extracted from the tumor tissues and normal colon tissues during operation and subjected to analysis of microsatellite instability (MSI) in 42 Chinese patients aged less than 50 with CRC. Mutation screenings were performed with denaturing high-performance liquid chromatography (DHPLC) followed by DNA sequencing of DNA samples with variant peaks, and genomic deletion detection with quantitative multiplex PCR (Q-M-PCR) in the patients uncovered with MSI(+). RESULTS: 22 out of the 42 (52.4%) patients investigated were microsatellite instability positive (MSI(+)), 10/42 MSI(+)-H and 12/42 MSI(+)-L. 8 kinds of DNA germline alterations, 5 polymorphisms and 3 novel point mutations, were found in 9 patients with MSI(+). A large DNA (exon 1-6) deletion in MSH2 gene and a missense mutation Met242Ile in MLH1 gene were unveiled in CRC tissues of two patients with MSI(+)-H. CONCLUSION: Mutations of mismatch repair genes are frequent in Chinese patients of CRC with onset at early ages.