BACKGROUND: In the repair of massive tissue defects using expanded large skin flaps, the incidence of complications increases with the size of the expanded area. Currently, stem cell therapy has limitations to solve this problem. We hypothesized that conditioned medium of adipose-derived stem cells (ADSC-CM) collected following mechanical pretreatment can assist skin expansion.
METHODS: Rat aortic endothelial cells and fibroblasts were cultured with ADSC-CM collected under 0%, 10%, 12%, and 15% stretching force. Ten-milliliter cylindrical soft tissue expanders were subcutaneously implanted into the backs of 36 Sprague-Dawley rats. The 0% and 10% stretch groups were injected with ADSC-CM collected under 0% and 10% stretching force, respectively, while the control group was not injected. After 3, 7, 14, and 30 days of expansion, expanded skin tissue was harvested for staining and qPCR analyses.
RESULTS: Endothelial cells had the best lumen formation and highest migration rate, and fibroblasts secreted the most collagen upon culture with ADSC-CM collected under 10% stretching force. The skin expansion rate was significantly increased in the 10% stretch group. After 7 days of expansion, the number of blood vessels in the expanded area, expression of the angiogenesis-associated proteins vascular endothelial growth factor, basic fibroblast growth factor, and hepatocyte growth factor, and collagen deposition were significantly increased in the 10% stretch group.
CONCLUSIONS: The optimal mechanical force upregulates specific paracrine proteins in ADSCs to increase angiogenesis and collagen secretion, and thereby promote skin regeneration and expansion. This study provides a new auxiliary method to expand large skin flaps.

Neurofibromatosis type 1 (NF1) is caused by NF1 gene mutations. Patients with NF1 often have complications with tumors, such as neurofibroma. In order to investigate the pathogenesis of human neurofibroma, a systematic comparison of protein expression levels between Schwann cell-like sNF96.2 cells, which originated from malignant peripheral nerve sheath tumors (MPNST), and normal Schwann cells was performed using 4-D label-free proteomic analysis. In addition, the expression levels and localization of dysregulated proteins were confirmed using a Gene Expression Omnibus (GEO) transcriptomic dataset, Western blot analysis, and immunofluorescence labeling. The effects of SRY-box transcription factor 9 (SOX9) in the neurofibroma and surrounding microenvironment were evaluated in vivo using a tumor transplantation model. The present study observed that SOX9 and procollagen C-endopeptidase enhancer (PCOLCE) were significantly altered. NF1 mutation promoted the nuclear translocation and transcriptional activity of SOX9 in neurofibromas. SOX9 increased collagen VI secretions by enhancing the activation of PCOLCE in neurofibroma cells. These findings might provide new perspectives on the pathophysiological significance of SOX9 in neurofibromas and elucidate a novel molecular mechanism underlying neurofibromas.

The diverse reproductive strategies of elasmobranchs (sharks, rays, and skates) have attracted research attention, but the endocrine control of reproduction is still incompletely known in elasmobranchs. By long-term monitoring of the egg-laying cycle in cloudy catsharks (Scyliorhinus torazame), we recently demonstrated a transient increase in plasma progesterone (P4) levels just prior to the appearance of the capsulated eggs in the oviducts. In the present study, we examined the in vivo effects of P4 administration in mature female cloudy catsharks. Although no capsulated eggs were observed following the implantation of P4-containing silicone tubing, we did find dark swollen oviducts in the abdominal cavity, in which clumps of long and coiled tendrils were observed. The tendril is an extension of the egg capsule, and the formation of the egg capsule begins with the tendril before main capsule formation. During the period of P4 implantation, the tendrils elongated, and their diameters were significantly increased on day 2 of treatment. Tendril formation was also confirmed on the day of endogenous P4 surge. Tendrils were not formed in catsharks implanted with estradiol-17β or testosterone. Histological analysis of the oviducal gland revealed that P4 implantation induced the secretion of tendril materials from the secretory tubules in the baffle zone, while the tendril materials were stored in the cytoplasm of the secretory cells under low P4 condition. Morphometrically, the area of secreted luminal materials in the secretory tubules was highly correlated to the diameter of tendrils. Our results suggest that the P4 surge during the egg-laying cycle serves as a trigger for egg capsule formation in the oviducal gland of cloudy catshark, but the hormonal signals were incomplete as the main capsule was not formed. Further studies are required to identify the hormones required for ovulation and formation of the main egg capsule.

Cosmetic products contain preservatives to prevent microbial growth. The various types of preservatives present in skincare products applied on the skin induce many side effects. We tested several types of preservatives such as phenoxyethanol, methyl paraben, propyl paraben, imidazolidinyl urea (IU), the composition of gluconolactone and sodium benzoate (GSB), diazolidinyl urea (DU), and two grapefruit essential oils, one of which was industrially produced and a second which was freshly distilled from fresh grapefruit peels. This study aimed to find the relationship between preservative concentration, cell growth, collagen secretion, and cell viability. We hypothesized that these products induced a decrease in collagen secretion from human dermal fibroblasts. Our research, for the first time, addressed the overall effect of other preservatives on skin extracellular matrix (ECM) by studying their effect on metalloproteinase-2 (MMP-2) activity. Except for cytotoxicity and contact sensitivity tests, there are no studies of their effect on skin ECM in the available literature. These studies show potential antimicrobial activity, especially from the compounds IU and DU towards reference bacteria and the compounds methyl paraben and propyl paraben against reference fungi. The MTS test showed that fibroblasts are more sensitive to the tested group of preservatives than keratinocytes, which could be caused by the differences between the cells\' structures. The grapefruit oils exhibited the most cytotoxicity to both tested cell lines compared to all considered preservatives. The most destructive influence of preservatives on collagen synthesis was observed in the case of IU and DU. In this case, the homemade grapefruit oil turned out to be the mildest one. The results from a diverse group of preservatives show that whether they are natural or synthesized compounds, they require controlled use. Appropriate dosages and evaluation of preservative efficacy should not be the only aspects considered. The complex effect of preservatives on skin processes and cytotoxicity is an important topic for modern people.

Plasma factor XIII (pFXIII) is a heterotetramer of FXIII-A and FXIII-B subunits. The cellular form (cFXIII), a dimer of FXIII-A, is present in a number of cell types. Activated FXIII (FXIIIa), a transglutaminase, plays an important role in clot stabilization, wound healing, angiogenesis and maintenance of pregnancy. It has a direct effect on vascular endothelial cells and fibroblasts, which have been implicated in the development of atherosclerotic plaques. Our aim was to explore the effect of FXIIIa on human aortic smooth muscle cells (HAoSMCs), another major cell type in the atherosclerotic plaque. Osteoblastic transformation induced by Pi and Ca2+ failed to elicit the expression of cFXIII in HAoSMCs. EZ4U, CCK-8 and CytoSelect Wound Healing assays were used to investigate cell proliferation and migration. The Sircol Collagen Assay Kit was used to monitor collagen secretion. Thrombospondin-1 (TSP-1) levels were measured by ELISA. Cell-associated TSP-1 was detected by the immunofluorescence technique. The TSP-1 mRNA level was estimated by RT-qPCR. Activated recombinant cFXIII (rFXIIIa) increased cell proliferation and collagen secretion. In parallel, a 67% decrease in TSP-1 concentration in the medium and a 2.5-fold increase in cells were observed. TSP-1 mRNA did not change significantly. These effects of FXIIIa might contribute to the pathogenesis of atherosclerotic plaques.

TANGO1 (transport and Golgi organization-1 homolog) encodes a transmembrane protein, which is located at endoplasmic reticulum (ER) exit sites where it binds bulky cargo, such as collagens, in the lumen and recruits membranes from the ER-Golgi intermediate compartment (ERGIC) to create an export route for cargo secretion. Mice lacking Mia3 (murine TANGO1 orthologue) show defective secretion of numerous procollagens and lead to neonatal lethality due to insufficient bone mineralization. Recently, aberrant expression of truncated TANGO1 in humans has been shown to cause a mild-to-moderate severe collagenopathy associated with dentinogenesis imperfecta, short stature, skeletal abnormalities, diabetes mellitus, and mild intellectual disability. We now show for the first time that complete loss of TANGO1 results in human embryonic lethality with near-total bone loss and phenocopies the situation of Mia3 -/- mice. Whole-exome sequencing on genomic DNA (gDNA) of an aborted fetus of Indian descent revealed a homozygous 4-base pair (4-bp) deletion in TANGO1 that is heterozygously present in both healthy parents. Parental fibroblast studies showed decreased TANGO1 mRNA expression and protein levels. Type I collagen secretion and extracellular matrix organization were normal, supporting a threshold model for clinical phenotype development. As such, our report broadens the phenotypic and mutational spectrum of TANGO1-related collagenopathies, and underscores the crucial role of TANGO1 for normal bone development, of which deficiency results in a severe-to-lethal form of osteochondrodysplasia. © 2021 American Society for Bone and Mineral Research © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

Porous zirconia (ZrO2), magnesia (MgO) and zirconia/magnesia (ZrO2/MgO) ceramics were synthesised by sintering and designated as ZrO2(100), ZrO2(75)MgO(25), ZrO2(50)MgO(50), ZrO2(25)MgO(75), MgO(100) based on their composition. The ceramic samples were characterised by means of scanning electron microscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy and atomic absorption spectrometry to explore the incorporation of Mg atoms into the zirconia lattice. The resulting porosity of the samples was calculated based on the composition and density. The final porosity of the cylinder-shaped ceramic samples ranged between 30 and 37%. The mechanical analysis exhibited that the Young modulus increased and the microstress decreased with increasing magnesia amount, with values ranging from 175 GPa for zirconia to 301 GPa for magnesia. The adhesion, viability, proliferation and osteogenic activity of MC3T3-E1 pre-osteoblastic cells cultured on the zirconia/magnesia ceramics was found to increase, with the magnesia-containing ceramics exhibiting higher values of calcium mineralisation. The results from the mechanical analysis, the ALP activity, the calcium and collagen production demonstrate that the zirconia/magnesia ceramics possess robust osteoinductive capacity, therefore holding great potential for bone tissue engineering.

BACKGROUND: Mutations in the SLC26A2 gene cause a spectrum of currently incurable human chondrodysplasias. However, genotype-phenotype relationships of SLC26A2-deficient chondrodysplasias are still perplexing and thus stunt therapeutic development.
METHODS: To investigate the causative role of SLC26A2 deficiency in chondrodysplasias and confirm its skeleton-specific pathology, we generated and analyzed slc26a2-/- and Col2a1-Cre; slc26a2fl/fl mice. The therapeutic effect of NVP-BGJ398, an FGFR inhibitor, was tested with both explant cultures and timed pregnant females.
RESULTS: Two lethal forms of human SLC26A2-related chondrodysplasias, achondrogenesis type IB (ACG1B) and atelosteogenesis type II (AO2), are phenocopied by slc26a2-/- mice. Unexpectedly, slc26a2-/- chondrocytes are defective for collagen secretion, exhibiting intracellular retention and compromised extracellular deposition of ColII and ColIX. As a consequence, the ATF6 arm of the unfolded protein response (UPR) is preferentially triggered to overactivate FGFR3 signaling by inducing excessive FGFR3 in slc26a2-/- chondrocytes. Consistently, suppressing FGFR3 signaling by blocking either FGFR3 or phosphorylation of the downstream effector favors the recovery of slc26a2-/- cartilage cultures from impaired growth and unbalanced cell proliferation and apoptosis. Moreover, administration of an FGFR inhibitor to pregnant females shows therapeutic effects on pathological features in slc26a2-/- newborns. Finally, we confirm the skeleton-specific lethality and pathology of global SLC26A2 deletion through analyzing the Col2a1-Cre; slc26a2fl/fl mouse line.
CONCLUSIONS: Our study unveils a previously unrecognized pathogenic mechanism underlying ACG1B and AO2, and supports suppression of FGFR3 signaling as a promising therapeutic approach for SLC26A2-related chondrodysplasias. FUND: This work was supported by National Natural Science Foundation of China (81871743, 81730065 and 81772377).

BACKGROUND: Recently, protein from ginseng was studied and used for the treatment of several kinds of diseases. However, the effect of ginseng total protein (GTP) on proliferation and wound healing in fibroblast cells remains unclear.
METHODS: In this study, cell viability was analyzed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Cell cycle distribution was analyzed by flow cytometer. The levels of transforming growth factor β1, vascular endothelial growth factor, and collagens were analyzed by enzyme-linked immunosorbent assay and immunofluorescence staining. The expressions of cyclin A, phosphorylation of extracellular signal-related kinase (p-ERK1/2), and ERK1/2 were analyzed by Western blotting.
RESULTS: Our results showed that GTP promoted cell proliferation and increased the percentage of cells in S phase through the upregulation of cyclin A in NIH/3T3 cells. We also found that GTP induced the secretion of type I collagen, and promoted the expression of other factors that regulate the synthesis of collagen such as transforming growth factor β1 and vascular endothelial growth factor. In addition, the phosphorylation of ERK1/2 at Thr202/Tyr204 was also increased by GTP.
CONCLUSIONS: Our studies suggest that GTP promoted proliferation and secretion of collagen in NIH/3T3 cells by activating the ERK signal pathway, which shed light on a potential function of GTP in promoting wound healing.

Hepatic stellate cells (HSCs) play an important role in liver fibrosis. This study investigates the expression of hedgehog in HSC and the role of hedgehog signaling on activation and collagen secretion of HSC. Liver ex vivo perfusion with collagenase IV and density gradient centrifugation were used to isolate HSC. Expression of hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 in HSC were detected by RT-PCR. Hedgehog siRNA vectors targeting Ihh, Smo and Gli2 were constructed and transfected into HSC respectively. Suppression of hedgehog signaling were detected by SYBR Green fluorescence quantitative RT-PCR. Effects of hedgehog signaling inhibition on HSC activation and collagen I secretion were analyzed. Hedgehog signaling components Ihh, Smo, Ptc, Gli2 and Gli3 were expressed in HSC. siRNA vectors targeting Ihh, Smo and Gli2 were successfully constructed and decreased target gene expression. Suppression of hedgehog signaling significantly decreased the expression of α-SMA in HSC (P<0.01). Collagen type I secretion of HSC were also significantly decreased (P<0.01). In summary, HSC activation and collagen secretion can be regulated by hedgehog signaling. Hedgehog may play a role in the pathogenesis of liver fibrosis.