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ABSTRACT The discovery of severe acute respiratory syndrome-coronavirus-2-like and Middle East respiratory syndrome-coronavirus-like viruses in Malayan pangolins has raised concerns about their potential role in the spread of zoonotic diseases. Herein, we describe the isolation and whole-genome sequencing of potentially zoonotic two bacterial pathogens from diseased Malaysian pangolins (Manis javanica)—Corynebacterium ulcerans and Erysipelothrix sp. The newly identified species were designated as C. ulcerans P69 and Erysipelothrix sp. P66. C. ulcerans P69 exhibited 99.2% whole-genome nucleotide identity to human bacterial isolate 4940, suggesting that it might have zoonotic potential. Notably, C. ulcerans P69 lacked the diphtheria toxin (tox) gene that is widely used in vaccines to protect humans from corynebacterial infection, which suggests that the current vaccine may be of limited efficacy against this pangolin strain. C. ulcerans P69 also contains other known virulence-associated genes such as pld and exhibits resistance to several antibiotics (erythromycin, clindamycin, penicillin G, gentamicin, tetracycline), which may affect its effective control. Erysipelothrix sp. P66 was closely related to Erysipelothrix sp. strain 2-related strains, exhibiting 98.8% whole-genome nucleotide identity. This bacterium is lethal in mice, and two commercial vaccines failed to protect its challenge, such that it could potentially pose a threat to the swine industry. Overall, this study highlights that, in addition to viruses, pangolins harbor bacteria that may pose a potential threat to humans and domestic animals, and which merit attention.IMPORTANCEThis study firstly reports the presence of two potentially zoonotic bacteria, Corynebacterium ulcerans and Erysipelothrix sp., in diseased Malaysian pangolins collected in 2019. The pangolin C. ulcerans is lethal in mice and resists many antibiotics. It clustered with a lethal human strain but lacked the diphtheria toxin gene. Diphtheria toxin is widely used as a vaccine around the world to protect humans from the infection of corynebacteria. The lack of the tox gene suggests that the current vaccine may be of limited efficacy against this pangolin strain. The pangolin Erysipelothrix sp. is the sister clade of Erysipelothrix rhusiopathiae. It is lethal in mice, and two commercial vaccines failed to protect the mice against challenge with the pangolin Erysipelothrix sp., such that this strain could potentially pose a threat to the swine industry. These findings emphasize the potential threat of pangolin bacteria.

BackgroundSmall extracellular vesicles derived from mesenchymal stem cells (MSC-sEVs) have emerged as a promising therapy for treating type II diabetic cutaneous wounds. Currently, the evidence supporting the use of MSC-sEVs for treating diabetic skin wounds remains inconclusive and is limited to preclinical studies. To facilitate the clinical translation of cell-free therapy, conducting a comprehensive systematic review of preclinical studies assessing the efficacy of MSC-sEVs is imperative.MethodsA systematic search was conducted on PubMed, Web of Science, Embase, and Cochrane Library databases until June 14, 2023, to identify studies that met our pre-established inclusion criteria. The outcome indicators comprised wound closure rate (primary outcome), neovascular density, re-epithelialization rate, collagen deposition, and inflammatory factors (secondary Outcomes). A fixed-effects model was employed in instances of low heterogeneity (I2

Diabetic neuropathy affects nearly half of all diabetics and poses a significant threat to public health. Recent preclinical studies suggest that mesenchymal stem cells (MSCs) may represent a promising solution for the treatment of diabetic neuropathy. However, an objective assessment of the preclinical effectiveness of MSCs is still pending. We conducted a comprehensive search of PubMed, Web of Science, Embase, and Cochrane library to identify preclinical studies that investigate the effects of MSCs on diabetic neuropathy up until 15 September 2023. Outcome indicators consisted of motor and sensory nerve conduction velocities, intra-epidermal nerve fiber density, sciatic nerve blood flow, capillary-to-muscle fiber ratio, neurotrophic factors, angiogenic factors and inflammatory cytokines. The literature review and meta-analysis were conducted independently by two researchers. 23 studies that met the inclusion criteria were included in this system review for qualitative and quantitative analysis. Pooled analyses indicated that MSCs exhibited an evident benefit in diabetic neuropathy in terms of motor (SMD = 2.16, 95% CI: 1.71–2.61) and sensory nerve conduction velocities (SMD = 2.93, 95% CI: 1.78–4.07), intra-epidermal nerve fiber density (SMD = 3.17, 95% CI: 2.28–4.07), sciatic nerve blood flow (SMD = 2.02, 95% CI: 1.37–2.66), and capillary-to-muscle fiber ratio (SMD = 2.28, 95% CI: 1.55 to 3.01, p < 0.00001). Furthermore, after MSC therapy, the expressions of neurotrophic and angiogenic factors increased significantly in most studies, while the levels of inflammatory cytokines were significantly reduced. The relevance of this review relies on the fact that summarizes an extensive body of work entailing substantial preclinical evidence that supports the efficacy of MSCs in mitigating diabetic neuropathy. While MSCs emerge as a promising potential treatment for diabetic neuropathy, further research is essential to elucidate the underlying mechanisms and the best administration strategy for MSCs.

A total of 151 recombinant inbred lines (RILs) were derived from the cross between ‘Cucumis sativus L. hardwickii’ (HW) and a cultivated Northern Chinese inbred line ‘XinTaiMiCi’ (XTMC). We used resequencing to construct the genetic map and analyze the genetic background of RIL population, and combined with the phenotypes of RIL population and the analysis of RNA-seq data, we located the major loci controlling the fruit length of cucumber and related analysis. A genetic map containing 600 bin markers was constructed via re-sequencing. Based on the phenotype data collected in two different seasons (spring 2021 and autumn 2022), the major quantitative trait loci (QTLs) controlling cucumber fruit length were located and their transcriptomic analysis carried out. The results revealed three QTLs (Fl2.1, Fl4.1, and Fl6.1) detected repeatedly in the two seasons, of which Fl4.1 was the dominant QTL. From the functional annotation of corresponding genes there, we discovered the gene Csa4G337340 encoding an auxin efflux carrier family protein. The expression of that gene was significantly lower in XTMC and the long-fruit RIL lines than in HW and the short-fruit RIL lines; hence, we speculated the gene could be negatively correlated with the fruit length of cucumber. Transcriptomic analysis showed that 259 differentially expressed genes (DEGs) were enriched in the plant hormone signal transduction pathway. In addition, among those DEGs, 509 transcription factors were detected, these distributed in several transcription factor gene families, such as bHLH, AP2/ErF -ERF, C2H2, and NAC. Therefore, we concluded that the major gene controlling the fruit length of cucumber is located in the interval of Fl4.1, whose gene Csa4G337340 may be involved in the negative regulation of fruit length. Further, genes related to plant hormone signal transduction and several transcription factors were also found involved in the regulation of cucumber fruit length. Our results provide a reference for the fine mapping of major genes and analyzing the mechanism of cucumber fruit length.

Ginger has been reported to potentially treat Alzheimer’s disease (AD), but the specific compounds responsible for this biological function and their mechanisms are still unknown. In this study, a combination of network pharmacology, molecular docking, and dynamic simulation technology was used to screen active substances that regulate AD and explore their mechanisms. The TCMSP, GeneCards, OMIM, and DisGeNET databases were utilized to obtain 95 cross-targets related to ginger’s active ingredients and AD as key targets. A functional enrichment analysis revealed that the pathways in which ginger’s active substances may be involved in regulating AD include response to exogenous stimuli, response to oxidative stress, response to toxic substances, and lipid metabolism, among others. Furthermore, a drug-active ingredient–key target interaction network diagram was constructed, highlighting that 6-Gingerol is associated with 16 key targets. Additionally, a protein–protein interaction (PPI) network was mapped for the key targets, and HUB genes (ALB, ACTB, GAPDH, CASP3, and CAT) were identified. Based on the results of network pharmacology and cell experiments, 6-Gingerol was selected as the active ingredient for further investigation. Molecular docking was performed between 6-Gingerol and its 16 key targets, and the top three proteins with the strongest binding affinities (ACHE, MMP2, and PTGS2) were chosen for molecular dynamics analysis together with the CASP3 protein as the HUB gene. The findings indicate that 6-Gingerol exhibits strong binding ability to these disease targets, suggesting its potential role in regulating AD at the molecular level, as well as in abnormal cholinesterase metabolism and cell apoptosis, among other related regulatory pathways. These results provide a solid theoretical foundation for future in vitro experiments using actual cells and animal experiments to further investigate the application of 6-Gingerol.

Single-view cone-beam X-ray luminescence computed tomography (CB-XLCT) has recently gained attention as a highly promising imaging technique that allows for the efficient and rapid three-dimensional visualization of nanophosphor (NP) distributions in small animals. However, the reconstruction performance is hindered by the ill-posed nature of the inverse problem and the effects of depth variation as only a single view is acquired. To tackle this issue, we present a methodology that integrates an automated restarting strategy with depth compensation to achieve reconstruction. The present study employs a fast proximal gradient descent (FPGD) method, incorporating L0 norm regularization, to achieve efficient reconstruction with accelerated convergence. The proposed approach offers the benefit of retrieving neighboring multitarget distributions without the need for CT priors. Additionally, the automated restarting strategy ensures reliable reconstructions without the need for manual intervention. Numerical simulations and physical phantom experiments were conducted using a custom CB-XLCT system to demonstrate the accuracy of the proposed method in resolving adjacent NPs. The results showed that this method had the lowest relative error compared to other few-view techniques. This study signifies a significant progression in the development of practical single-view CB-XLCT for high-resolution 3−D biomedical imaging.

Tissue-engineered cartilage regeneration by bone marrow stromal cells (BMSCs) is considered an ideal method. However, how to regulate BMSCs to regenerate specific types of cartilage remains unclear, which significantly limits its clinical translation and leads to suboptimal clinical effects. Herein, we systematically explored the role of native ear and articular cartilage niches on the differentiation fate of BMSCs and the type of regenerated cartilage. First, we prepared two types of acellular cartilage sheets (ACSs) and two types of chondrocytes. Then green fluorescent protein-labeled BMSCs were seeded on two types of ACSs with or without corresponding types of chondrocytes using a sandwich model and directed or cross-implanted them into native cartilage niches. After one year of in vivo culture, cell tracking and the results of histological results showed that the native cartilage niches were capable of regulating BMSCs regeneration into specific types of cartilage that were consistent with the cartilage types of the implanted sites. Furthermore, even when the type of niche formed by ACSs or the biomimetic cartilage niche constructed by specific types of ACSs and specific types of chondrocytes did not match with the native cartilage niche, the native cartilage niche continued to determine the type of cartilage regenerated by implanted BMSCs and chondrocytes. All our results provide sufficient evidence for specific types of cartilage regeneration using chondrogenic potential cells, such as mesenchymal stem cells and chondrocytes.

Purpose: To develop the safest possible environment for treating urgent patients with COVID-19 requiring radiation, we describe the unique construction of negative air pressure computed tomography simulator and linear accelerator treatment vaults in addition to screening, delay, and treatment protocols and their evolution over the course of the COVID-19 pandemic. Methods and Materials: Construction of large high-efficiency particulate air filter air-flow systems into existing ductwork in computed tomography simulator rooms and photon and proton treatment vaults was completed to create negative-pressure rooms. An asymptomatic COVID-19 screening protocol was implemented for all patients before initiation of treatment. Patients could undergo simulation and/or treatment in the biocontainment environments according to a predefined priority scale and protocol. Patients treated under the COVID-19 protocol from June 2020 to January 2022 were retrospectively reviewed. Results: Negative air-flow environments were created across a regional network, including a multi-gantry proton therapy unit. In total, 6525 patients were treated from June 2020 through January 2022 across 5 separate centers. The majority of patients with COVID-19 had radiation treatment deferred when deemed safe. A total of 42 patients with COVID-19, who were at highest risk of an adverse outcome should there be a radiation delay, were treated under the COVID-19 biocontainment protocol in contrast to those who were placed on treatment break. For 61.9% of patients, these safety measures mitigated an extended break during treatment. The majority of patients (64.3%) were treated with curative intent. The median number of biocontainment sessions required by each patient was 6 (range, 1-15) before COVID-19 clearance and resumption of treatment in a normal air-flow environment. Conclusions: Constructing negative-pressure environments and developing a COVID-19 biocontainment treatment protocol allowed for the safe treatment of urgent radiation oncology patients with COVID-19 within our department and strengthens future biopreparedness. These biocontainment units set a high standard of safety in radiation oncology during the current or for any future infectious outbreak.

In this paper, an off-set fin heat exchanger (OSFHE) and a flying-wing fin heat exchanger (FWFHE) were developed for the condenser of a condensing clothes dryer. They were used to replace the slit fin heat exchanger (SFHE), which is the prototype condenser. Performance parameters, like condensation rate, IEC energy efficiency index (EEI) and specific moisture extraction rate (SMER), were used to assess the dehumidification performance of the heat exchangers and the drying performance of the dryer. Though the fin areas of OSFHE and FWFHE are 20% and 34.2% less than that of SFHE, the experiment results indicated that OSFHE and FWFHE had a faster condensation rate than SFHE during the main drying stage. And the heat exchanger with a faster condensation rate also led to less power consumption of the dryer in most tests. Compared with using SFHE, using OSFHE could decrease the EEI of the dryer by 0.43%. Using FWFHE could reduce the EEI of the dryer by 1.19% and improve the EU energy efficiency class of the dryer from Class C to Class B. Both OSFHE and FWFHE are able to replace SFHE and become a new generation of condensers for clothes dryers.

Abstract Background We hypothesized that the total volume of metastases at initial oligometastatic (OM) presentation to stereotactic body radiation therapy (SBRT) is an important prognostic factor that can refine the definition of OM disease. Methods Patients with extracranial oligometastatic cancer (≤5 lesions) treated with SBRT were included in an international multi‐institutional database. Multivariable Cox and competing risks regression models were used to determine the relationship between distant progression‐free survival (DPFS), widespread progression (WSP), and overall survival (OS) with the total planning target volume (PTV) at initial OM presentation to SBRT. All models were adjusted for histology, pre‐SBRT systemic therapy, osseous‐only lesions, and number of metastases. Results In total, 961 patients were included. The median follow‐up was 24.4 months (IQR: 13.8–37.5). Total PTV had a significant effect on DPFS in the first 18 months after SBRT and was most profound in the first 6 months, when each twofold increase in total PTV conferred a 40.6% increased risk of distant progression (p

Abstract Autologous bone marrow mononuclear cell (BMMNC) transplantation has been widely studied in recent years. The fresh cell cocktail in BMMNCs, without going through the in vitro culture process, helps to establish a stable microenvironment for osteogenesis, and each cell type may play a unique role in bone regeneration. Our study compared the efficacy of concentrated fresh BMMNCs and cultured bone marrow‐derived mesenchymal stem cells (BMSCs) in Beagle dogs for the first time. Fifteen‐millimeter segmental bone defects were created in the animals' tibia bones. In BMMNCs group, the defects were repaired with concentrated fresh BMMNCs combined with β‐TCP (n = 5); in cultured BMSC group, with in vitro cultured and osteo‐induced BMSCs combined with β‐TCP (n = 5); in scaffold‐only group, with a β‐TCP graft alone (n = 5); and in blank group, nothing was grafted (n = 3). The healing process was monitored by X‐rays and single photon emission computed tomography. The animals were sacrificed 12 months after surgery and their tibias were harvested and analyzed by microcomputed tomography and hard tissue histology. Moreover, the microstructure, chemical components, and microbiomechanical properties of the regenerated bone tissue were explored by multiphoton microscopy, Raman spectroscopy and nanoindentation. The results showed that BMMNCs group promoted much more bone regeneration than cultured BMSC group. The grafts in BMMNCs group were better mineralized, and they had collagen arrangement and microbiomechanical properties similar to the contralateral native tibia bone. These results indicate that concentrated fresh bone marrow mononuclear cells may be superior to in vitro expanded stem cells in segmental bone defect repair.

The introduction of the N-oxide functional group to the heterocyclic structure is deemed as an effective strategy to construct highly energetic and lowly sensitive materials. Nevertheless, the detailed influence of N-oxidation on the detonation performance and sensitive properties of energetic materials (EMs) is still fragmentary. Herein, a total of 34 experimental known crystals of energetic N-oxides and their analogues were screened, and the detonation performances along with sensitivities were comparatively analyzed to reveal the role of N-oxidation. The results show that crystal density and packing coefficients increase as the introduction of N-oxide groups in most cases, leading to high detonation performances. Compared with analogues, conversely, the decomposition temperatures and sensitivities of energetic N-oxide decrease and rise, respectively, indicative of poor safety. Hence, it is difficult to both promote energy and safety by N-oxidation relative to the analogues. However, the introduction of N-oxide groups is still helpful for the creation of novel EMs since some energetic N-oxides can possess high energy and low sensitivity comparable to HMX. Additionally, several theoretical approaches of sensitivity estimation were tested and inconsistent prediction results were obtained, demonstrating that sensitivity prediction only at the molecular level is not enough for N-oxide systems.

For the various imbalances of series-connected cells, the existing balancing methods has the disadvantages of limited energy flow direction and unnecessary charge transfer. Based on flyback converter, this paper proposes a multi-winding input and multi-winding output bi-directional equalization topology. It can realize any cell (s) to any cell (s) balancing, which has the advantage of flexible balancing energy flow path. This paper adopts the control strategy of time-sharing participation in equalization according to the order of the initial state of charge (SOC) of cells, it can solve the problem of unnecessary energy transfer and simplify the control logic. In addition, it gets rid of the influence of energy loss of the circuit components on the setting of the fixed equalization end judgment value, whether equalization ends or not depends on the difference between the discharge cells and the charge cells in this paper, which has the advantage of random adaptive adjustment. In this paper, the operational principle of the proposed circuit are analyzed and the equalization strategy is described in detail. A comparison in balancing performance between the conventional equalization topology based on flyback converter and proposed topology is shown in static state. To verify the validity of the proposed method, the simulation experiment of four cells series-connected is carried out, and the simulation results show that the balancing method proposed in this paper has faster equalization speed and higher equalization energy transfer efficiency.

Cell–hydrogel constructs are frequently used as injectable platforms for irregular cartilage regeneration. However, cell–hydrogel constructs have obvious disadvantages, such as long culture times, high probability of infection, and poor cartilage formation capacity, significantly limiting their clinical translation. In this study, we aimed to develop a novel injectable platform comprising engineered cartilage gel (ECG) and gelatin methacrylate (GelMA) to improve cartilage regeneration. We first prepared an ECG by cutting the in vitro engineered cartilage sheet into pieces. The chondrocytes and ECG were evenly encapsulated into GelMA to form Cell-GelMA and ECG-GelMA constructs. The ECG-GelMA construct exhibited preferred gel characteristics and superior biocompatibility compared with the Cell-GelMA construct counterpart. After subcutaneous implantation in nude mice and goat, both gross views and histological evaluations showed that the ECG-GelMA construct achieved more homogenous, stable, and mature cartilage regeneration than the Cell-GelMA construct. Immunological evaluations showed that ECG-GelMA had a mitigatory immunologic reaction than the Cell-GelMA construct. Overall, the results suggest that the ECG-GelMA is a promising injectable platform for cartilage regeneration that may advance clinical translation.

Tissue engineering provides a new approach for the treatment of osteochondral defects. However, the lack of an ideal double-layer scaffold with osteochondral-biomimetic microenvironment and interface similar to native articular tissue greatly limits clinical translation. Our current study developed a double-layer acellular osteochondral matrix (AOM) scaffold with natural osteochondral-biomimetic microenvironment and interface by integrating ultraviolet (UV) laser and decellularization techniques. The laser parameters were optimized to achieve a proper pore depth close to the osteochondral interface, which guaranteed complete decellularization, sufficient space for cell loading, and relative independence of the chondrogenic and osteogenic microenvironments. Gelatin-methacryloyl (GelMA) hydrogel was further used as the cell carrier to significantly enhance the efficiency and homogeneity of cell loading in the AOM scaffold with large pore structure. Additionally, in vitro results demonstrated that the components of the AOM scaffold could efficiently regulate the chondrogenic/osteogenic differentiations of bone marrow stromal cells (BMSCs) by activating the chondrogenic/osteogenic related pathways. Importantly, the AOM scaffolds combined with BMSC-laden GelMA hydrogel successfully realized tissue-specific repair of the osteochondral defects in a knee joint model of rabbit. The current study developed a novel double-layer osteochondral biomimetic scaffold and feasible strategy, providing strong support for the tissue-specific repair of osteochondral defects and its future clinical translation.

Abstract Background Dai, Hani, and Yao people, in the trans-boundary region between China, Laos, and Vietnam, have gathered plentiful traditional knowledge about wild edible plants during their long history of understanding and using natural resources. The ecologically rich environment and the multi-ethnic integration provide a valuable foundation and driving force for high biodiversity and cultural diversity in this region. However, little study has uncovered this unique and attractive culture to the world. Methods We conducted ethnobotanical survey in 20 villages of Jiangcheng County from 2016 to 2020. Altogether 109 local Dai, Hani, and Yao people were interviewed, and their traditional knowledge about wild edible plants was recorded. Voucher specimens were identified by the authors and deposited in the herbarium of Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences (HITBC). The use value was used as a quantitative index to evaluate the consumption frequency and relative importance of the wild edible plants. The Jaccard index was calculated to assess the usage similarity of different areas. The relationship of age and recognized wild edible plants by different ethnic people was performed by R. Results A total of 211 wild edible plants, belonging to 71 families and 151 genera, were recorded. These plants were consumed as wild edible vegetables, seasonal fruits, salads, spices, sour condiments, tonic soups, tea substitutes, liquor brewing, or dyeing materials. The use value (UV), current cultivation, market availability, and the quantitative traditional knowledge inheritance situation of these wild edible plants among different generations, were analyzed. Based on the data from the threatened species list of China’s higher plants and the IUCN Red List, the food plant list for Asia Elephant, the Subject Database of China Plant, and the calculated UV score, the top 30 most important wild edible plants were selected for further cultivation in some local villages. Conclusion Traditional knowledge of wild edible plants, owned by Dai, Hani, and Yao people in Jiangcheng County, is rich but at risk of being lost among the young generation. Diversified cultivation of wild edible plants by the local communities could be a solution for the sustainable use of natural resources and to conserve the endangered species in this trans-boundary region.

Abstracts: Crystal packing modes determine the energy and safety performance of energetic materials (EMs) to some extent. To deepen the understanding of the intermolecular interactions in the crystal packings, the homologous molecular crystals of energetic materials were first defined according to the packing similarities in this study, and four groups of energetic homologous crystals with the same packing structures were found from the large experimentally available data. Several computational approaches were employed to comparatively probe the intermolecular interactions at molecular and crystal levels. The results of the analysis show that the common organic skeleton of two molecular conformations in each homologous packing structure remains the same or becomes mirror-symmetrical with a minor variation of functional groups, resulting in the local change of molecular electrostatic potentials, while the overall distribution of electrostatic potentials can be largely maintained. Compared with the other groups of homologous crystals, interestingly, the differences in intermolecular interactions in Group 4 are relatively large according to two-dimensional fingerprint plots and energy frameworks, indicating that the crystal packing of energetic materials is predominately caused by some types of intermolecular interactions. The results reported here are expected to inspire the constructions of energetic crystals with specific packing modes to improve performances, such as a minor modification to the molecular structure while keeping original crystal packing style.

Although cartilage regeneration technology has achieved clinical breakthroughs, whether auricular chondrocytes (AUCs) represent optimal seed cells to achieve stable cartilage regeneration is not clear. In this study, we systematically explore biological behaviors of human- and goat-derived AUCs during in vitro expansion as well as cartilage regeneration in vitro and in vivo. To eliminate material interference, a cell sheet model was used to evaluate the feasibility of dedifferentiated AUCs to re-differentiate and regenerate cartilage in vitro and in vivo. We found that the dedifferentiated AUCs could re-differentiate and regenerate cartilage sheets under the chondrogenic medium system, and the generated chondrocyte sheets gradually matured with increased in vitro culture time (2, 4, and 8 weeks). After the implantation of cartilage sheets with different in vitro culture times in nude mice, optimal neocartilage was formed in the group with 2 weeks in vitro cultivation. After in vivo implantation, ossification only occurred in the group with goat-regenerated cartilage sheet of 8 weeks in vitro cultivation. These results, which were confirmed in human and goat AUCs, suggest that AUCs are ideal seed cells for the clinical translation of cartilage regeneration under the appropriate culture system and culture condition.

Abstract Fat grafting procedures are considered to be a promising regenerative, cell‐directed therapy; however, their survival is mainly influenced by ischemia condition. Fructose 1,6‐bisphosphate (FBP), as an intermediate in energy metabolism, has the potential to rescue cells and tissues from hypoxic‐ischemic circumstances. In the present study, human lipoaspirates were grafted subcutaneously into nude mice followed by a daily intraperitoneal injection of FBP at different doses for 7 days. Next, the grafts were harvested at different time points till 12 weeks postimplantation and were evaluated for cell viability and function, tissue revascularization and inflammatory cell infiltration using histological analysis, whole‐mount living tissue imaging, glycerol 3‐phosphate dehydrogenase activity assays, and quantitative analysis of gene expression. The results demonstrated that exogenous FBP administration could attenuate the volume and weight reduction of fat graft; meanwhile, FBP enhanced adipocyte viability and function, increased blood vessel formation, and decreased inflammation. Moreover, in vitro cell experiments showed that FBP could promote adipose‐derived stem cell viability and vascular endothelial growth factor (VEGF) mRNA expression in ischemia conditions. Our study indicates that FBP can be used as a protective agent for fat grafting and may be applied in stem cell‐based regenerative medicine. Stem Cells Translational Medicine 2019;8:606–616

In vitro construction is a major trend involved in cartilage regeneration and repair. Satisfactory in vitro cartilage regeneration depends on a suitable culture system. Current chondrogenic culture systems with a high content of transforming growth factor beta-1 effectively promote cartilaginous extracellular matrix (ECM) production but inhibit chondrocyte survival. As is known, inhibition of the c-Jun N-terminal kinase (JNK) signaling pathway acts in blocking the progression of osteoarthritis by reducing chondrocyte apoptosis and cartilage destruction. However, whether inhibiting JNK signaling resists the inhibitory effect of current chondrogenic medium (CM) on cell survival and affects in vitro auricular cartilage regeneration (including cell proliferation, ECM synthesis, and degradation) has not been investigated. In order to address these issues and optimize the chondrogenic culture system, we generated a three-dimensional in vitro auricular cartilage regeneration model to investigate the effects of SP600125 (a JNK-specific inhibitor) on chondrocyte proliferation and ECM metabolism. SP600125 supplementation efficiently promoted cell proliferation at both cellular and tissue levels and canceled the negative effect of our chondrogenic culture system on cell survival. Moreover, it significantly inhibited ECM degradation by reducing the expressions of tumor necrosis factor-alpha, interleukin-1-beta, and matrix metalloproteinase 13. In addition, SP600125 inhibited ECM synthesis at both cellular and tissue levels, but this could be canceled and even reversed by adding chondrogenic factors; yet this enabled a sufficient number of chondrocytes to be retained at the same time. Thus, SP600125 had a positive effect on in vitro auricular cartilage regeneration in terms of cell proliferation and ECM degradation but a negative effect on ECM synthesis, which could be reversed by adding CM. Therefore, a combination of SP600125 and CM might help in optimizing current chondrogenic culture systems and achieve satisfactory in vitro cartilage regeneration by promoting cell proliferation, reducing ECM degradation, and enhancing ECM synthesis.

Scaffold-free cartilage-sheet technology can stably regenerate high-quality cartilage tissue in vivo. However, uncontrolled shape maintenance and mechanical strength greatly hinder its clinical translation. Decalcified bone matrix (DBM) has high porosity, a suitable pore structure, and good biocompatibility, as well as controlled shape and mechanical strength. In this study, cartilage sheet was prepared into engineered cartilage gel (ECG) and combined with DBM to explore the feasibility of regenerating 3D cartilage with controlled shape and mechanical strength. The results indicated that ECG cultured in vitro for 3 days (3 d) and 15 days (15 d) showed good biocompatibility with DBM, and the ECG–DBM constructs successfully regenerated viable 3D cartilage with typical mature cartilage features in both nude mice and autologous goats. Additionally, the regenerated cartilage had comparable mechanical properties to native cartilage and maintained its original shape. To further determine the optimal seeding parameters for ECG, the 3 d ECG regenerated using human chondrocytes was diluted in different concentrations (1:3, 1:2, and 1:1) for seeding and in vivo implantation. The results showed that the regenerated cartilage in the 1:2 group exhibited better shape maintenance and homogeneity than the other groups. The current study established a novel mode of 3D cartilage regeneration based on the design concept of steel (DBM)-reinforced concrete (ECG) and successfully regenerated homogenous and mature 3D cartilage with controlled shape and mechanical strength, which hopefully provides an ideal cartilage graft for the repair of various cartilage defects.

An acellular matrix (AM) as a kind of natural biomaterial is gaining increasing attention in tissue engineering applications. An acellular cartilaginous matrix (ACM) and acellular dermal matrix (ADM) are two kinds of the most widely used AMs in cartilage tissue engineering. However, there is still debate over which of these AMs achieves optimal cartilage regeneration, especially in immunocompetent large animals. In the current study, we fabricated porous ADM and ACM scaffolds by a freeze-drying method and confirmed that ADM had a larger pore size than ACM. By recolonization with goat auricular chondrocytes and in vitro culture, ADM scaffolds exhibited a higher cell adhesion rate, more homogeneous chondrocyte distribution, and neocartilage formation compared with ACM. Additionally, quantitative polymerase chain reaction (qPCR) indicated that expression of cartilage-related genes, including ACAN, COLIIA1, and SOX9, was significantly higher in the ADM group than the ACM group. Furthermore, after subcutaneous implantation in a goat, histological evaluation showed that ADM achieved more stable and matured cartilage compared with ACM, which was confirmed by quantitative data including the wet weight, volume, and contents of DNA, GAG, total collagen, and collagen II. Additionally, immunological assessment suggested that ADM evoked a low immune response compared with ACM as evidenced by qPCR and immunohistochemical analyses of CD3 and CD68, and TUNEL. Collectively, our results indicate that ADM is a more suitable AM for cartilage regeneration, which can be used for cartilage regeneration in immunocompetent large animals.

Inspired by the natural photosynthetic system in which proteins control the electron transfer from electron donors to acceptors, in this research, artificial polymers were tried to achieve this control effect. Polyvinylpyrrolidone (PVP) was found to form complex with pigments 5,10,15,20-tetrakis-(4-sulfonatophenyl) porphyrin (TPPS) and its zinc complex (ZnTPPS) quantitatively through different interactions (hydrogen bonds and coordination bonds, respectively). These complex formations hinder the interaction between ground-state TPPS or ZnTPPS and an electron acceptor (methyl viologen, MV2+) and could control the photoinduced electron transfer from TPPS or ZnTPPS to MV2+, giving more electron transfer products methyl viologen cationic radical (MV+•). Other polymers such as PEG did not show similar results, indicating that PVP plays an important role in controlling the photoinduced electron transfer.

Abstract Background At present, many laboratories and hospitals all over the world are attempting and exploring the clinical transformation of this tissue engineered bone graft (TEBG) strategy. Many successful cases of bone tissue engineering (BTE) repair were based on young individuals. But there are little studies about the effectiveness of TEBG strategy in physiological aged individuals. Methods In this research, we studied whether aging factor has influence on the skull repair effect of Fetal-TEBG, at the level of the large animal models. We used the fetal bone marrow stromal cells (Fetal-BMSCs) as the seed cells, combining the decalcified bone matrix (DBM) scaffolds, to repair the skull defects of the aged goats and the young goats. The repair effects on both aged goat and young goat were compared by Micro-CT and histology examination. Results The skull defects of the young goats could be repaired better than that of the aged goats after 6 months by Fetal-TEBG; In the aged goats, although not completely repaired, the defects repaired by Fetal-TEBG was better than that repaired by the Control DBM scaffold. Conclusions Aging factor has impact on the bone repair effect of Fetal-TEBG; and the BTE strategy is still efficacious even in the aged individuals. The improvement of the aged state may promote the repair effect of the BTE in the aged individuals.

Abstract Background Bone tissue engineering is not widely used in clinical treatment. Two main reasons hide behind this: (1) the seed cells are difficult to obtain and (2) the process of tissue engineering bone construction is too complex and its efficiency is still relatively low. It is foreseeable that in the near future, the problem of seed cell sources could be solved completely in tissue engineering bone repair. As for the complex process and low efficiency of tissue engineering bone construction, usually two strategies would be considered: (1) the construction strategy based on injectable bone tissue and (2) the construction strategy based on osteogenic cell sheets. However, the application of injectable bone tissue engineering (iBTE) strategy and osteogenic cell sheet strategy is limited and they could hardly be used directly in repairing defects of large segmental bone, especially load-bearing bone. Methods In this study, we built an osteogenic micro-tissue with simple construction but with a certain structure and composition. Based on this, we established a new iBTE repair strategy—osteogenic micro-tissue in situ repair strategy, mainly targeting at solving the problem of large segmental bone defect. The steps are as follows: (1) Build the biodegradable three-dimensional scaffold based on the size of the defect site with 3D printing rapid prototyping technology. (2) Implant the three-dimensional scaffold into the defect site. This scaffold is considered as the “steel framework” that could provide both mechanical support and space for bone tissue growth. (3) Inject the osteogenic micro-tissue (i.e., the “cell-extracellular matrix” complex), which could be considered as “concrete,” into the three-dimensional scaffold, to promote the bone tissue regeneration in situ. Meanwhile, the digested cells were injected as the compared group in this experiment. After 3 months, the effect of in situ bone defect repair of osteogenic micro-tissue and digested cells was compared. Results It is confirmed that osteogenic micro-tissue could achieve a higher efficiency on cell usage and has a better repair effect than the digested cells. Conclusions Osteogenic micro-tissue repairing strategy would be a more promising clinical strategy to solve the problem of large segmental bone defect.

Purpose: For patients with localized pancreatic cancer (PC) with vascular involvement, prediction of resectability is critical to define optimal treatment. However, the current definitions of borderline resectable (BR) and locally advanced (LA) disease leave considerable heterogeneity in outcomes within these classifications. Moreover, factors beyond vascular involvement likely affect the ability to undergo resection. Herein, we share our experience developing a model that incorporates detailed radiologic, patient, and treatment factors to predict surgical resectability in patients with BR and LA PC who undergo stereotactic body radiation therapy (SBRT). Methods and materials: Patients with BR or LA PC who were treated with SBRT between 2010 and 2016 were included. The primary endpoint was margin negative resection, and predictors included age, sex, race, treatment year, performance status, initial staging, tumor volume and location, baseline and pre-SBRT carbohydrate antigen 19-9 levels, chemotherapy regimen and duration, and radiation dose. In addition, we characterized the relationship between tumors and key arteries (superior mesenteric, celiac, and common hepatic arteries), using overlap volume histograms derived from computed tomography data. A classification and regression tree was built, and leave-one-out cross-validation was performed. Prediction of surgical resection was compared between our model and staging in accordance with the National Comprehensive Care Network guidelines using McNemar’s test. Results: A total of 191 patients were identified (128 patients with LA and 63 with BR), of which 87 patients (46%) underwent margin negative resection. The median total dose was 33 Gy. Predictors included the chemotherapy regimen, amount of arterial involvement, and age. Importantly, radiation dose that covers 95% of gross tumor volume (GTV D95), was a key predictor of resectability in certain subpopulations, and the model showed improved accuracy in the prediction of margin negative resection compared with National Comprehensive Care Network guideline staging (75% vs 63%; P < .05). Conclusions: We demonstrate the ability to improve prediction of surgical resectabiliy beyond the current staging guidelines, which highlights the value of assessing vascular involvement in a continuous manner. In addition, we show an association between radiation dose and resectability, which suggests the potential importance of radiation to allow for resection in certain populations. External data are needed for validation and to increase the robustness of the model.

Microtia is a congenital external ear malformation that can seriously influence the psychological and physiological well-being of affected children. The successful regeneration of human ear-shaped cartilage using a tissue engineering approach in a nude mouse represents a promising approach for auricular reconstruction. However, owing to technical issues in cell source, shape control, mechanical strength, biosafety, and long-term stability of the regenerated cartilage, human tissue engineered ear-shaped cartilage is yet to be applied clinically. Using expanded microtia chondrocytes, compound biodegradable scaffold, and in vitro culture technique, we engineered patient-specific ear-shaped cartilage in vitro. Moreover, the cartilage was used for auricle reconstruction of five microtia patients and achieved satisfactory aesthetical outcome with mature cartilage formation during 2.5 years follow-up in the first conducted case. Different surgical procedures were also employed to find the optimal approach for handling tissue engineered grafts. In conclusion, the results represent a significant breakthrough in clinical translation of tissue engineered human ear-shaped cartilage given the established in vitro engineering technique and suitable surgical procedure. This study was registered in Chinese Clinical Trial Registry (ChiCTR-ICN-14005469).

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engineering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered cartilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.

The regeneration of tissue‐engineered cartilage in an immunocompetent environment usually fails due to severe inflammation induced by the scaffold and their degradation products. In the present study, we compared the tissue remodeling and the inflammatory responses of engineered cartilage constructed with bone marrow mesenchymal stem cells (BMSCs), chondrocytes, or both and scaffold group in pigs. The cartilage‐forming capacity of the constructs in vitro and in vivo was evaluated by histological, biochemical, and biomechanical analyses, and the inflammatory response was investigated by quantitative analysis of foreign body giant cells and macrophages. Our data revealed that BMSC‐based engineered cartilage suppressed in vivo inflammation through the alteration of macrophage phenotype, resulting in better tissue survival compared with those regenerated with chondrocytes alone or in combination with BMSCs. To further confirm the macrophage phenotype, an in vitro coculture system established by engineered cartilage and macrophages was studied using immunofluorescence, enzyme‐linked immunosorbent assay, and gene expression analysis. The results demonstrated that BMSC‐based engineered cartilage promoted M2 polarization of macrophages with anti‐inflammatory phenotypes including the upregulation of CD206, increased IL‐10 synthesis, decreased IL‐1β secretion, and alterations in gene expression indicative of M1 to M2 transition. It was suggested that BMSC‐seeded constructs have the potential to ameliorate scaffold‐induced inflammation and improve cartilaginous tissue regeneration through M2 polarization of macrophages. Significance Finding a strategy that can prevent scaffold‐induced inflammation is of utmost importance for the regeneration of tissue‐engineered cartilage in an immunocompetent environment. This study demonstrated that bone marrow mesenchymal stem cell (BMSC)‐based engineered cartilage could suppress inflammation by increasing M2 polarization of macrophages, resulting in better tissue survival in a pig model. Additionally, the effect of BMSC‐based cartilage on the phenotype conversion of macrophages was further studied through an in vitro coculture system. This study could provide further support for the regeneration of cartilage engineering in immunocompetent animal models and provide new insight into the interaction of tissue‐engineered cartilage and macrophages.

Adipose-derived stem cells (ASCs) can be applied extensively in the clinic because they can be easily isolated and cause less donor-site morbidity; however, their application can be complicated by patient-specific factors, such as age and harvest site. In this study, we systematically evaluated the effects of age on the quantity and quality of human adipose-derived mesenchymal stem cells (hASCs) isolated from excised chest subcutaneous adipose tissue and investigated the underlying molecular mechanism. hASCs were isolated from donors of 3 different age-groups (i.e., child, young adult, and elderly). hASCs are available from individuals across all age-groups and maintain mesenchymal stem cell (MSC) characteristics. However, the increased age of the donors was found to have a significant negative effect on hASCs frequency base on colony-forming unit fibroblasts assay. Moreover, there is a decline in both stromal vascular fraction (SVF) cell yield and the proliferation rate of hASCs with increasing age, although this relationship is not significant. Aging increases cellular senescence, which is manifested as an increase in SA-β-gal-positive cells, increased mitochondrial-specific reactive oxygen species (ROS) production, and the expression of p21 in the elderly. Further, advancing age was found to have a significant negative effect on the adipogenic and osteogenic differentiation potentials of hASCs, particularly at the early and mid-stages of induction, suggesting a slower response to the inducing factors of hASCs from elderly donors. Finally, impaired migration ability was also observed in the elderly group and was determined to be associated with decreased expression of chemokine receptors, such as CXCR4 and CXCR7 . Taken together, these results suggest that, while hASCs from different age populations are phenotypically similar, they present major differences at the functional level. When considering potential applications of hASCs in cell-based therapeutic strategies, the negative influence of age on hASC differentiation potential and migration abilities should be taken seriously.

Cytokine-induced killer cells (CIK) have been used in clinic for adoptive immunotherapy in a variety of malignant tumors and have improved the prognosis of cancer patients. However, there are individual differences in the CIK cell preparations including the obvious differences in the ratio of effector CIK cells among different cancer patients. Infusion of such heterogeneous immune cell preparation is an important factor that would affect the therapeutic efficacy. We report here the enrichment and expansion of CD8+ cells from CIK cells cultured for one week using magnetic activated cell sorting (MACS). These enriched CD8+ CIK cells expressed T cell marker CD3 and antigen recognition receptor NKG2D. Phenotypic analysis showed that CD8+ CIK cells contained 32.4% of CD3+ CD56+ natural killer (NK)-like T cells, 23.6% of CD45RO+ CD28+, and 50.5% of CD45RA+ CD27+ memory T cells. In vitro cytotoxic activity assay demonstrated that the enriched CD8+ CIK cells had significant cytotoxic activity against K562 cells and five ovarian cancer cell lines. Intriguingly, CD8+ CIK cells had strong cytotoxic activity against OVCAR3 cells that has weak binding capability to NKG2D. Flow cytometry and quantitative RT-PCR analysis revealed that OVCAR3 cells expressed HLA-I and OCT4 and Sox2, suggesting that CD8+ CIK cells recognize surface antigen via specific T cell receptor and effectively kill the target cells. The results suggest that transplantation of such in vitro enriched and expanded OCT4-specific CD8+ CIK cells may improve the specific immune defense mechanism against cancer stem cells, providing a novel avenue of cancer stem cell targeted immunotherapy for clinical treatment of ovarian cancer.

The goal of this study is to determine the effects of Insulin-Transferrin-Selenium (ITS) on proliferation of auricular chondrocytes and formation of engineered cartilage in vitro. Pig auricular monolayer chondrocytes and chondrocyte pellets were cultured in media containing 1% ITS at different concentrations of fetal bovine serum (FBS, 10%, 6%, 2%, 0%), or 10% FBS alone as a control for four weeks. Parameters including cell proliferation in monolayer, wet weight, collagen type I/II/X (Col I, II, X) and glycosaminoglycan (GAG) expression, GAG content of pellets and gene expression associated with cartilage formation/dedifferentiation (lost cartilage phenotype)/hypertrophy within the chondrocyte pellets were assessed. The results showed that chondrocytes proliferation rates increased when FBS concentrations increased (2%, 6%, 10% FBS) in ITS supplemented groups. In addition, 1% ITS plus 10% FBS significantly promoted cell proliferation than 10% FBS alone. No chondrocytes grew in ITS alone medium. 1% ITS plus 10% FBS enhanced cartilage formation in terms of size, wet weight, cartilage specific matrices, and homogeneity, compared to 10% FBS alone group. Furthermore, ITS prevented engineered cartilage from dedifferentiation (i.e., higher index of Col II/Col I mRNA expression and expression of aggrecan) and hypertrophy (i.e., lower mRNA expression of Col X and MMP13). In conclusion, our results indicated that ITS efficiently enhanced auricular chondrocytes proliferation, retained chondrogenic phenotypes, and promoted engineered cartilage formation when combined with FBS, which is potentially used as key supplementation in auricular chondrocytes and engineered cartilage culture.

Allogeneic bone marrow mesenchymal stem cell (allo-BMSC)-based tissue-engineered bone (TEB) has great potential for bone defect repair. However, the immunogenicities and biological roles of allo-BMSCs are still controversial. In this study, we established an animal model of critical-sized mandibular defect in beagle dogs and compared the repairing effects of allo-BMSC-based TEB with autogenic BMSC (auto-BMSC)-based TEB without the administration of immunosuppressants. During the first 2 weeks postimplantation, a transient immune response in the allo-BMSC group was detected with an increase in proinflammation cytokines TNF-α, IFN-γ, and IL-2, a declination of anti-inflammation cytokine IL-10, and an increase in percentages of CD4 + and CD8 + T-cell subsets in peripheral blood. Nevertheless, there was no significant difference in bone union achievement, bone mineral density, and biomechanical properties between the two groups at 12 and 24 weeks postimplantation. Further subcutaneous implantation of allo-BMSCs/scaffold also exhibited the similar transient immune responses in the first 2 weeks postimplantation but followed by a decreased bone formation at 4 and 8 weeks postimplantation. These findings indicate that allo-BMSCs can induce a transient immunoreaction, which may temporally delay the osteogenesis of allo-BMSC/scaffold complex in early stage of in vivo implantation, whereas the long-term engineered bone formation was not affected.

Although the feasibility of human bone marrow mesenchymal stem cell (hBMMSC)-based tissue-engineered bone (TEB) has been proven in a number of studies, reaching a high positive fraction and bone yield of TEB still remains a challenge. Here we report a dose–effect relationship of the quantity of seeded cells with in vivo bone yield and the required quantity of hBMMSCs for the effective, stable bone formation of TEB. In our study, TEB was constructed using the static seeding technique with the gradient of seeding densities and volumes of passage 3 hBMMSCs. The in vitro characteristics of seeding efficiency, proliferation, viability, distribution, and osteogenic differentiation of hBMMSCs seeded on two commercial scaffolds of β-TCP and CHA were investigated using alamarBlue assay, live/dead staining, confocal laser scanning microscope, scanning electronic microscopy examination, and mRNA expression analysis of osteogenic differentiation markers. After 3 months of ectopic implantation, in vivo bone regeneration was examined by quantitative analysis of histology and micro-CT. The results showed that 10 × 10 6 cells/ml was the minimum cell seeding density for CHA and β-TCP to generate new bone in vivo. In addition, 20 × 10 6 cells/ml and 30 × 10 6 cells/ml were the saturating seeding densities for CHA and β-TCP to produce new bone effectively and stably, respectively. Thus, for different scaffolds, the saturating seeding density should be investigated first to ensure the effectiveness and stability of TEB construction with minimum donor injury, which is essential for the clinical application of TEB.

Acellular corneal stroma matrix has been used for corneal stroma engineering. However, because of its compact tissue structure, regrowth of keratocytes into the scaffold is difficult. Previously, we developed a sandwich model for cartilage engineering using acellular cartilage sheets. In the present study, we tested this model for corneal stroma regeneration using acellular porcine corneal stroma (APCS) sheets and keratocytes. Porcine corneas were decellularized by NaCl treatment, and the APCS was cut into 20-μm-thick sheets. A rabbit corneal stroma defect model was created by lamellar keratoplasty and repaired by transplantation of five pieces of APCS sheets with keratocytes. Six months after transplantation, transparent corneas were present in the experimental group, which were confirmed by anterior segment optical coherence tomography examination and transmittance examination. The biomechanical properties in the experimental group were similar to those of normal cornea. Histological analyses showed an even distribution of keratocytes and well-oriented matrix in the stroma layer in the experimental group. Together, these results demonstrated that the sandwich model using acellular corneal stroma sheets and keratocytes could be potentially useful for corneal stroma regeneration.

Endothelial progenitor cells (EPCs) from bone marrow have proven to be functional for the prevention of liver fibrosis in chronic liver injury. However, expansion of EPCs in culture is complicated and expansive. Previously, we have established a simple method that could enrich and expand EPCs by simple seeding bone marrow cells in high density dots. The purpose of this study is to evaluate whether cells derived from high-density (HD) culture of rat bone marrow cells could prevent the liver fibrosis in a chronic liver injury rat model, induced by carbon tetrachloride (CCl4). Flow cytometric analysis showed that cells from HD culture were enriched for EPCs, expressing high levels of EPC markers. Intrasplenic injection of HD cultured bone marrow cells in the CCl4-induced liver injury rat showed an enhanced antifibrogenic effect compared with animals treated with cells from regular-density culture. The antifibrogenic effect was demonstrated by biochemical and histological analysis 4 weeks post-transplantation. Furthermore, cells from HD culture likely worked through increasing neovascularization, stimulating liver cell proliferation, and suppressing pro-fibrogenic factor expression. HD culture, which is a simple and cost-effective procedure, could potentially be used to expand bone marrow cells for the treatment of liver fibrosis.

In vitro expansion of endothelial progenitor cells (EPCs) remains a challenge in stem cell research and its application. We hypothesize that high density culture is able to expand EPCs from bone marrow by mimicking cell-cell interactions of the bone marrow niche. To test the hypothesis, rat bone marrow cells were either cultured in high density (2 × 10(5) cells/cm(2)) by seeding total 9 × 10(5) cells into six high density dots or cultured in regular density (1.6 × 10(4) cells/cm(2)) with the same total number of cells. Flow cytometric analyses of the cells cultured for 15 days showed that high density cells exhibited smaller cell size and higher levels of marker expression related to EPCs when compared to regular density cultured cells. Functionally, these cells exhibited strong angiogenic potentials with better tubal formation in vitro and potent rescue of mouse ischemic limbs in vivo with their integration into neo-capillary structure. Global gene chip and ELISA analyses revealed up-regulated gene expression of adhesion molecules and enhanced protein release of pro-angiogenic growth factors in high density cultured cells. In summary, high density cell culture promotes expansion of bone marrow contained EPCs that are able to enhance tissue angiogenesis via paracrine growth factors and direct differentiation into endothelial cells.

T lymphocytes (T cells) undergo metabolic reprogramming after activation to provide energy and biosynthetic materials for growth, proliferation and differentiation. Distinct T cell subsets, however, adopt metabolic programs specific to support their needs. As CD4 T cells coordinate adaptive immune responses while CD8 T cells become cytotoxic effectors, we compared activation-induced proliferation and metabolic reprogramming of these subsets. Resting CD4 and CD8 T cells were metabolically similar and used a predominantly oxidative metabolism. Following activation CD8 T cells proliferated more rapidly. Stimulation led both CD4 and CD8 T cells to sharply increase glucose metabolism and adopt aerobic glycolysis as a primary metabolic program. Activated CD4 T cells, however, remained more oxidative and had greater maximal respiratory capacity than activated CD8 T cells. CD4 T cells were also associated with greater levels of ROS and increased mitochondrial content, irrespective of the activation context. CD8 cells were better able, however, to oxidize glutamine as an alternative fuel source. The more glycolytic metabolism of activated CD8 T cells correlated with increased capacity for growth and proliferation, along with reduced sensitivity of cell growth to metabolic inhibition. These specific metabolic programs may promote greater growth and proliferation of CD8 T cells and enhance survival in diverse nutrient conditions.