Fibromodulin Reduces Scar Size and Increases Scar Tensile Strength in Normal and Excessive-Mechanical-Loading Porcine Cutaneous Wounds

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive-mechanical-loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule—fibromodulin (FMOD) protein—can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive-mechanical-loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD-based technologies hold high translational potential and applicability to human patients suffering from scarring—especially hypertrophic scarring. © 2018 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine

Delayed Wound Closure in Fibromodulin-Deficient Mice Is Associated with Increased TGF-β3 Signaling

Fibromodulin (FMOD), a small leucine-rich proteoglycan, mediates scarless fetal skin wound repair through, in part, transforming growth factor-Β (TGF-Β) modulation. Using an adult fmod-null (fmod -/-) mouse model, this study further elucidates the interplay between FMOD and TGF-Β expression during cutaneous repair and scar formation. Full-thickness skin wounds on fmod -/- and wild-type (WT) mice were closed primarily and analyzed. Histomorphometry revealed delayed dermal cell migration leading to delayed wound closure and significantly increased scar size in fmod -/- mice relative to WT, which was partially rescued by exogenous FMOD administration. In addition, fmod -/- wounds exhibited early elevation (within 24 hours post-wounding) of type I and type II TGF-Β receptors as well as unexpectedly high fibroblast expression of TGF-Β3, a molecule with reported antifibrotic and antimigratory effects. Consistent with elevated fibroblastic TGF-Β3, fmod -/- fibroblasts were significantly less motile than WT fibroblasts. fmod -/- fibroblasts were also more susceptible to migration inhibition by TGF-Β3, leading to profound delays in dermal cell migration. Increased scarring in fmod -/- mice indicates that TGF-Β3\u27s antimotility effects predominate over its antifibrotic effects when high TGF-Β3 levels disrupt early fibroblastic wound ingress. These studies demonstrate that FMOD presence is critical for proper temporospatial coordination of wound healing events and normal TGF-Β bioactivity. © 2011 The Society for Investigative Dermatology

Use of human perivascular stem cells for bone regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering(1-3). PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines(1,2). In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)(8). FSDs are bicortical and are stabilized with a polyethylene bar and K-wires(4). The FSD described is also a critical size defect, which does not significantly heal on its own(4). In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models

Human Perivascular Stem Cells Show Enhanced Osteogenesis and Vasculogenesis with Nel-Like Molecule I Protein

An ideal mesenchymal stem cell (MSC) source for bone tissue engineering has yet to be identified. Such an MSC population would be easily harvested in abundance, with minimal morbidity and with high purity. Our laboratories have identified perivascular stem cells (PSCs) as a candidate cell source. PSCs are readily isolatable through fluorescent-activated cell sorting from adipose tissue and have been previously shown to be indistinguishable from MSCs in the phenotype and differentiation potential. PSCs consist of two distinct cell populations: (1) pericytes (CD146+, CD34−, and CD45−), which surround capillaries and microvessels, and (2) adventitial cells (CD146−, CD34+, and CD45−), found within the tunica adventitia of large arteries and veins. We previously demonstrated the osteogenic potential of pericytes by examining pericytes derived from the human fetal pancreas, and illustrated their in vivo trophic and angiogenic effects. In the present study, we used an intramuscular ectopic bone model to develop the translational potential of our original findings using PSCs (as a combination of pericytes and adventitial cells) from human white adipose tissue. We evaluated human PSC (hPSC)-mediated bone formation and vascularization in vivo. We also examined the effects of hPSCs when combined with the novel craniosynostosis-associated protein, Nel-like molecule I (NELL-1). Implants consisting of the demineralized bone matrix putty combined with NELL-1 (3 μg/μL), hPSC (2.5×10(5) cells), or hPSC+NELL-1, were inserted in the bicep femoris of SCID mice. Bone growth was evaluated using microcomputed tomography, histology, and immunohistochemistry over 4 weeks. Results demonstrated the osteogenic potential of hPSCs and the additive effect of hPSC+NELL-1 on bone formation and vasculogenesis. Comparable osteogenesis was observed with NELL-1 as compared to the more commonly used bone morphogenetic protein-2. Next, hPSCs induced greater implant vascularization than the unsorted stromal vascular fraction from patient-matched samples. Finally, we observed an additive effect on implant vascularization with hPSC+NELL-1 by histomorphometry and immunohistochemistry, accompanied by in vitro elaboration of vasculogenic growth factors. These findings hold significant implications for the cell/protein combination therapy hPSC+NELL-1 in the development of strategies for vascularized bone regeneration

The Nell-1 Growth Factor Stimulates Bone Formation by Purified Human Perivascular Cells

The search for novel sources of stem cells other than bone marrow mesenchymal stem cells (MSCs) for bone regeneration and repair has been a critical endeavor. We previously established an effective protocol to homogeneously purify human pericytes from multiple fetal and adult tissues, including adipose, bone marrow, skeletal muscle, and pancreas, and identified pericytes as a primitive origin of human MSCs. In the present study, we further characterized the osteogenic potential of purified human pericytes combined with a novel osteoinductive growth factor, Nell-1. Purified pericytes grown on either standard culture ware or human cancellous bone chip (hCBC) scaffolds exhibited robust osteogenic differentiation in vitro. Using a nude mouse muscle pouch model, pericytes formed significant new bone in vivo as compared to scaffold alone (hCBC). Moreover, Nell-1 significantly increased pericyte osteogenic differentiation, both in vitro and in vivo. Interestingly, Nell-1 significantly induced pericyte proliferation and was observed to have pro-angiogenic effects, both in vitro and in vivo. These studies suggest that pericytes are a potential new cell source for future efforts in skeletal regenerative medicine, and that Nell-1 is a candidate growth factor able to induce pericyte osteogenic differentiation

NELL-1, an osteoinductive factor, is a direct transcriptional target of Osterix.

NELL-1 is a novel secreted protein associated with premature fusion of cranial sutures in craniosynostosis that has been found to promote osteoblast cell differentiation and mineralization. Our previous study showed that Runx2, the key transcription factor in osteoblast differentiation, transactivates the NELL-1 promoter. In this study, we evaluated the regulatory involvement and mechanisms of Osterix, an essential transcription factor of osteoblasts, in NELL-1 gene expression and function. Promoter analysis showed a cluster of potential Sp1 sites (Sp1/Osterix binding sites) within approximately 70 bp (from -71 to -142) of the 5' flanking region of the human NELL-1 transcriptional start site. Luciferase activity in our NELL-1 promoter reporter systems was significantly decreased in Saos-2 cells when Osterix was overexpressed. Mutagenesis study demonstrated that this suppression is mediated by the Sp1 sites. The binding specificity of Osterix to these Sp1 sites was confirmed in Saos-2 cells and primary human osteoblasts by EMSA in vitro and ChIP assay in vivo. ChIP assay also showed that Osterix downregulated NELL-1 by affecting binding of RNA polymerase II to the NELL-1 promoter, but not by competing with Runx2 binding to the OSE2 sites. Moreover, NELL-1 mRNA levels were significantly decreased when Osterix was overexpressed in Saos-2, U2OS, Hela and Glioma cells. Correspondingly, knockdown of Osterix increased NELL-1 transcription and osteoblastic differentiation in both Saos-2 cells and primary human osteoblasts. These results suggest that Osterix is a direct transcriptional regulator with repressive effect on NELL-1 gene expression, contributing to a delicate balance of regulatory effects on NELL-1 transcription with Runx2, and may play a crucial role in osteoblast differentiation and mineralization. These findings also extend our understanding of the molecular mechanism of Runx2, Osterix, and NELL-1 and demonstrate their crosstalk during osteogenesis

Nell-1 Enhances Bone Regeneration in a Rat Critical-Sized Femoral Segmental Defect Model

Exterior detail of the “musical walls”; [The buildings contain a hundred sleeping rooms for teachers and students, study halls, a hall for work and one for recreation, a library and a refectory. There is also a church, where the monks practice, and the circulation, which connects all the parts (the achievement of the traditional cloister form is rendered impossible here by the slope of terrain)]. In La Tourette, the public spaces, particularly the central atrium, are stimulated by the rhythmic play of the lines of the “musical walls” or “pans de verre ondulatoire,” (designed by Xenakis) whereas the static concrete box of the adjacent church has vertical perforations that reach up behind the altar. The lateral chapels are animated with machine guns of light (mitraillettes à lumière) painted in vibrant colors in the crypt and the sacristy. In 1960, speaking of this convent, Le Corbusier evoked a theme that he had been working on since the 1940s: "When a work reaches its maximum level of intensity, proportion, quality of execution, and perfection, a phenomenon of ineffable space occurs: the places radiate, physically they radiate. They become what I call “ineffable space,” that is to say, an impact based not on dimensions but on perfection. This is about the ineffable domain." Source: Grove Art Online; http://www.oxfordartonline.com/ (accessed 5/7/2011

Osterix specifically binds to Sp1 sites in the <i>NELL-1</i> promoter <i>in vitro</i> and <i>in vivo.</i>

<p>(<b>A</b>) EMSA of Saos-2 nuclear proteins transfected with pCtr or pOsx binding to SiteA (containing three proximal Sp1 sites) and SiteB probes. Supershifts with specific Osterix antibody indicate the specific Osterix-DNA complexes. (<b>B</b>) EMSA depicting primary human osteoblast cell (hOB) nuclear proteins transfected with pOsx binding to the SiteA probes, with competition by 20x and 200x unlabeled SiteA and 200x unlabeled mutated SiteA (MutA) oligonucleotides. Note that the MutA probes failed to bind nuclear proteins. The same pattern is also seen by competition of SiteB element and MutB. (<b>C–D</b>) Osterix binds to endogenous Sp1 sites of the human <i>NELL-1</i> promoter in Saos-2 (C) and hOB (D) cells. The <i>NELL-1</i> -1 kb primer set covers 1 kb proximal promoter region containing all Sp1/Osterix binding sites and three OSE2 sites. The <i>NELL-1</i> -2 kb primer set covers <i>NELL-1</i> promoter region from −1 kb to −2 kb where no Sp1/Osterix binding site but one OSE2 site exists. The qPCR products depict DNA amplified from Chromatin Immunoprecipitation with cells utilizing Control IgG and Osterix antibody. Input DNA represents positive genomic DNA control.</p