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Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

ABSTRACT: Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

SUBMITTER: Han Q

PROVIDER: S-EPMC4529088 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

Han Qianqian Q Yang Pishan P Wu Yuwei Y Meng Shu S Sui Lei L Zhang Lan L Yu Liming L Tang Yin Y Jiang Hua H Xuan Dongying D Kaplan David L DL Kim Sung Hoon SH Tu Qisheng Q Chen Jake J

Tissue engineering. Part A 20150608 15-16

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal p ...[more]

PMID: 25923143

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Project description:Mesenchymal stromal cells (MSCs) have shown to be a promising tool in cell therapies to treat different conditions. Several pre-clinical and clinical studies have proved that the transplantation of MSCs improves wound healing. Here, we compare the beneficial effects of mouse bone marrow-derived allogeneic MSCs (allo-mBM-MSCs) and their acelullar derivatives (allo-acd-mMSCs) on skin wound healing in Non-Obese Diabetic (NOD) mice. One dose of allo-mBM-MSCs (1×106 cells) or one dose of allo-acd-mMSCs (1X) were intradermally injected around wounds in 8-10 week old female NOD mice. Wound healing was evaluated macroscopically (wound closure) every two days, and microscopically (reepithelialization, dermoepidermal junction, skin appendage regeneration, leukocyte infiltration, vascularization, granulation tissue formation, and density of collagen fibers in the dermis) after 16 days of MSC injection. In addition, we measured growth factors and specific proteins that were present in the allo-acd-mMSCs. Results showed significant differences in the wound healing kinetics of lesions that received allo-acd-mMSCs compared to lesions that received vehicle or allo-mBM-MSCs. In particular, mice treated with allo-acd-mMSCs reached significantly higher percentages of wound closure at day 4, 6 and 8, relative to the allo-mBM-MSCs and vehicle groups (p < 0.05), while wound closure percentages could not be statistically distinguished between the allo-mBM-MSCs and vehicle groups. Also, allo-acd-mMSCs had a greater influence in the skin would healing process. Specifically, they caused a less pronounced inflammatory severe response (p < 0.0001), more granulation tissue formation at an advanced stage (p < 0.0001), and higher density of collagen fibers (p < 0.05) compared to the other groups. Nevertheless, at day 16, both allo-mBM-MSCs and allo-acd-mMSCs revealed a higher effect on the recovery of the quality skin (continuous epidermis; regular dermoepidermal junction and skin appendages) relative to untreated lesions (p < 0.0001), but not between them. On the other hand, ELISA analyses indicated that the allo-acd-mMSCs contained growth factors and proteins relevant to wound healing such as IGF-1, KGF, HGF, VEGF, ANG-2, MMP-1, CoL-1 and PGE2. Compared to allo-acd-mMSCs, the administration of allo-mBM-MSCs is insufficient for wound healing in diabetic mice and delays the therapeutic effect, which maybe explained by the fact that trophic factors secreted by MSCs are critical for skin regeneration, and not the cells per se, suggesting that MSCs may require some time to secrete these factors after their administration.

Project description:Interfaces between soft tissue and bone are characterized by transitional gradients in composition and structure that mediate substantial changes in mechanical properties. For interfacial tissue engineering, scaffolds with mineral gradients have shown promise in controlling osteogenic behavior of seeded bone marrow stromal cells (bMSCs). Previously, we have demonstrated a 'top-down' method for creating monolithic bone-derived scaffolds with patterned mineral distributions similar to native tissue. In the present work, we evaluated the ability of these scaffolds to pattern osteogenic behavior in bMSCs in basic, osteogenic, and chondrogenic biochemical environments. Immunohistochemical (IHC) and histological stains were used to characterize cellular behavior as a function of local mineral content. Alkaline phosphatase, an early marker of osteogenesis, and osteocalcin, a late marker of osteogenesis, were positively correlated with mineral content in basic, osteogenic, and chondrogenic media. The difference in bMSC behavior between the mineralized and demineralized regions was most pronounced in an basic biochemical environment. In the mineralized regions of the scaffold, osteogenic markers were clearly present as early as 4 days in culture. In osteogenic media, osteogenic behavior was observed across the entire scaffold, whereas in chondrogenic media, there was an overall reduction in osteogenic biomarkers. Overall, these results indicate local mineral content of the scaffold plays a key role in spatially patterning bMSC behavior. Our results can be utilized for the development of interfacial tissue engineered scaffolds and understanding the role of local environment in determining bMSC behavior. STATEMENT OF SIGNIFICANCE: Soft tissue-to-bone interfaces, such as tendon-bone, ligament-bone, and cartilage-bone, are ubiquitous in mammalian musculoskeletal systems. These interfacial tissues have distinct, hierarchically-structured gradients of cellular, biochemical, and materials components. Given the complexity of the biological structures, interfacial tissues present unique challenges for tissue engineering. Here, we demonstrate that material-derived cues can spatially pattern osteogenic behavior in bone marrow stromal cells (bMSCs). Specifically, we observed that when the bMSCs are cultured on bone-derived scaffolds with mineral gradients, cells in contact with higher mineral content display osteogenic behavior at earlier times than those on the unmineralized substrate. The ability to pattern the cellular complexity found in native interfaces while maintaining biologically relevant structures is a key step towards creating engineered tissue interfaces.

Project description:Diabetes mellitus is a group of chronic diseases characterized by high blood glucose levels. Diabetic patients have a higher risk of sustaining osteoporotic fractures than non-diabetic people. The fracture healing is usually impaired in diabetics, and our understanding of the detrimental effects of hyperglycemia on fracture healing is still inadequate. Metformin is the first-line medicine for type 2 diabetes (T2D). However, its effects on bone in T2D patients remain to be studied. To assess the impacts of metformin on fracture healing, we compared the healing process of closed-wound fixed fracture, non-fixed radial fracture, and femoral drill-hole injury models in the T2D mice with and without metformin treatment. Our results demonstrated that metformin rescued the delayed bone healing and remolding in the T2D mice in all injury models. In vitro analysis indicated that compromised proliferation, osteogenesis, chondrogenesis of the bone marrow stromal cells (BMSCs) derived from the T2D mice were rescued by metformin treatment when compared to WT controls. Furthermore, metformin could effectively rescue the impaired detrimental lineage commitment of BMSCs isolated from the T2D mice in vivo as assessed by subcutaneous ossicle formation of the BMSC implants in recipient T2D mice. Moreover, the Safranin O staining of cartilage formation in the endochondral ossification under hyperglycemic condition significantly increased at day 14 post-fracture in the T2D mice receiving metformin treatment. The chondrocyte transcript factors SOX9 and PGC1α, important to maintain chondrocyte homeostasis, were both significantly upregulated in callus tissue isolated at the fracture site of metformin-treated MKR mice on day 12 post-fracture. Metformin also rescued the chondrocyte disc formation of BMSCs isolated from the T2D mice. Taken together, our study demonstrated that metformin facilitated bone healing, more specifically bone formation and chondrogenesis in T2D mouse models.

Project description:Recent work demonstrates that osteoprogenitor cell culture on nanofiber scaffolds can promote differentiation. This response may be driven by changes in cell morphology caused by the three-dimensional (3D) structure of nanofibers. We hypothesized that nanofiber effects on cell behavior may be mediated by changes in organelle structure and function. To test this hypothesis, human bone marrow stromal cells (hBMSCs) were cultured on poly(ε-caprolactone) (PCL) nanofibers scaffolds and on PCL flat spuncoat films. After 1 day-culture, hBMSCs were stained for actin, nucleus, mitochondria, and peroxisomes, and then imaged using 3D confocal microscopy. Imaging revealed that the hBMSC cell body (actin) and peroxisomal volume were reduced during culture on nanofibers. In addition, the nucleus and peroxisomes occupied a larger fraction of cell volume during culture on nanofibers than on films, suggesting enhancement of the nuclear and peroxisomal functional capacity. Organelles adopted morphologies with greater 3D-character on nanofibers, where the Z-Depth (a measure of cell thickness) was increased. Comparisons of organelle positions indicated that the nucleus, mitochondria, and peroxisomes were closer to the cell center (actin) for nanofibers, suggesting that nanofiber culture induced active organelle positioning. The smaller cell volume and more centralized organelle positioning would reduce the energy cost of inter-organelle vesicular transport during culture on nanofibers. Finally, hBMSC bioassay measurements (DNA, peroxidase, bioreductive potential, lactate, and adenosine triphosphate (ATP)) indicated that peroxidase activity may be enhanced during nanofiber culture. These results demonstrate that culture of hBMSCs on nanofibers caused changes in organelle structure and positioning, which may affect organelle functional capacity and transport. Published 2016. This article is a U.S. Government work and is in the public domain in the USA. J Biomed Mater Res Part B: Appl Biomater, 2016. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 989-1001, 2017.

Project description:IntroductionSilk fibroin (SF) scaffolds have been shown to be a suitable substrate for tissue engineering and to improve tissue regeneration when cellularized with mesenchymal stromal cells (MSCs). We here demonstrate, for the first time, that electrospun nanofibrous SF patches cellularized with human adipose-derived MSCs (Ad-MSCs-SF), or decellularized (D-Ad-MSCs-SF), are effective in the treatment of skin wounds, improving skin regeneration in db/db diabetic mice.MethodsThe conformational and structural analyses of SF and D-Ad-MSCs-SF patches were performed by scanning electron microscopy, confocal microscopy, Fourier transform infrared spectroscopy and differential scanning calorimetry. Wounds were performed by a 5 mm punch biopsy tool on the mouse's back. Ad-MSCs-SF and D-Ad-MSCs-SF patches were transplanted and the efficacy of treatments was assessed by measuring the wound closure area, by histological examination and by gene expression profile. We further investigated the in vitro angiogenic properties of Ad-MSCs-SF and D-Ad-MSCs-SF patches by affecting migration of human umbilical vein endothelial cells (HUVECs), keratinocytes (KCs) and dermal fibroblasts (DFs), through the aortic ring assay and, finally, by evaluating the release of angiogenic factors.ResultsWe found that Ad-MSCs adhere and grow on SF, maintaining their phenotypic mesenchymal profile and differentiation capacity. Conformational and structural analyses on SF and D-Ad-MSCs-SF samples, showed that sterilization, decellularization, freezing and storing did not affect the SF structure. When grafted in wounds of diabetic mice, both Ad-MSCs-SF and D-Ad-MSCs-SF significantly improved tissue regeneration, reducing the wound area respectively by 40% and 35%, within three days, completing the process in around 10 days compared to 15-17 days of controls. RT2 gene profile analysis of the wounds treated with Ad-MSCs-SF and D-Ad-MSCs-SF showed an increment of genes involved in angiogenesis and matrix remodeling. Finally, Ad-MSCs-SF and D-Ad-MSCs-SF co-cultured with HUVECs, DFs and KCs, preferentially enhanced the HUVECs' migration and the release of angiogenic factors stimulating microvessel outgrowth in the aortic ring assay.ConclusionsOur results highlight for the first time that D-Ad-MSCs-SF patches are almost as effective as Ad-MSCs-SF patches in the treatment of diabetic wounds, acting through a complex mechanism that involves stimulation of angiogenesis. Our data suggest a potential use of D-Ad-MSCs-SF patches in chronic diabetic ulcers in humans.

Dataset Information

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

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Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

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