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

Brief Review of Models of Ectopic Bone Formation

Ectopic bone formation is a unique biologic entity?distinct from other areas of skeletal biology. Animal research models of ectopic bone formation most often employ rodent models and have unique advantages over orthotopic (bone) environments, including a relative lack of bone cytokine stimulation and cell-to-cell interaction with endogenous (host) bone-forming cells. This allows for relatively controlled in vivo experimental bone formation. A wide variety of ectopic locations have been used for experimentation, including subcutaneous, intramuscular, and kidney capsule transplantation. The method, benefits and detractions of each method are summarized in the following review. Briefly, subcutaneous implantation is the simplest method. However, the most pertinent concern is the relative paucity of bone formation in comparison to other models. Intramuscular implantation is also widely used and relatively simple, however intramuscular implants are exposed to skeletal muscle satellite progenitor cells. Thus, distinguishing host from donor osteogenesis becomes challenging without cell-tracking studies. The kidney capsule (perirenal or renal capsule) method is less widely used and more technically challenging. It allows for supraphysiologic blood and nutrient resource, promoting robust bone growth. In summary, ectopic bone models are extremely useful in the evaluation of bone-forming stem cells, new osteoinductive biomaterials, and growth factors; an appropriate choice of model, however, will greatly increase experimental success.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98476/1/scd%2E2011%2E0517.pd

Fibromodulin-Deficiency Alters Temporospatial Expression Patterns of Transforming Growth Factor-β Ligands and Receptors during Adult Mouse Skin Wound Healing

<div><p>Fibromodulin (FMOD) is a small leucine-rich proteoglycan required for scarless fetal cutaneous wound repair. Interestingly, increased FMOD levels have been correlated with decreased transforming growth factor (TGF)-β1 expression in multiple fetal and adult rodent models. Our previous studies demonstrated that FMOD-deficiency in adult animals results in delayed wound closure and increased scar size accompanied by loose package collagen fiber networks with increased fibril diameter. In addition, we found that FMOD modulates <i>in vitro</i> expression and activities of TGF-β ligands in an <i>isoform-specific</i> manner. In this study, temporospatial expression profiles of TGF-β ligands and receptors in FMOD-null and wild-type (WT) mice were compared by immunohistochemical staining and quantitative reverse transcriptase-polymerase chain reaction using a full-thickness, primary intention wound closure model. During the inflammatory stage, elevated inflammatory infiltration accompanied by increased type I TGF-β receptor levels in individual inflammatory cells was observed in FMOD-null wounds. This increased inflammation was correlated with accelerated epithelial migration during the proliferative stage. On the other hand, significantly more robust expression of TGF-β3 and TGF-β receptors in FMOD-null wounds during the proliferative stage was associated with delayed dermal cell migration and proliferation, which led to postponed granulation tissue formation and wound closure and increased scar size. Compared with WT controls, expression of TGF-β ligands and receptors by FMOD-null dermal cells was markedly reduced during the remodeling stage, which may have contributed to the declined collagen synthesis capability and unordinary collagen architecture. Taken together, this study demonstrates that a single missing gene, <i>FMOD</i>, leads to conspicuous alternations in TGF-β ligand and receptor expression at all stages of wound repair in various cell types. Therefore, FMOD critically coordinates temporospatial distribution of TGF-β ligands and receptors <i>in vivo</i>, suggesting that FMOD modulates TGF-β bioactivity in a complex way beyond simple physical binding to promote proper wound healing.</p></div

Immunohistochemical (IHC) staining of wounded WT and FMOD-null adult mice skin.

<p>(<b>A</b>) TGF-β1, (<b>B</b>) TGF-β2, (<b>C</b>) TGF-β3, (<b>D</b>) TβRI, (<b>E</b>) TβRII, and (<b>F</b>) TβRIII. Inserts show low magnification view. Red arrowheads: inflammatory cells; open black triangles: epidermis at wound edge; solid black triangles: migrating epidermal tongues; blue arrows: dermal fibroblasts. Bar  = 100 µm.</p

Description of Primary Antibodies Used for Indirect Immunostaining.

<p>Description of Primary Antibodies Used for Indirect Immunostaining.</p

TaqMan® Gene Expression Assay.

<p>* Commercial TaqMan® Gene Expression Assays were purchased from Applied Biosystems, Foster City, CA, USA.</p

Hematoxylin and eosin (H&E) staining of wounded WT and FMOD-null adult mice skin.

<p>(<b>A</b>) At day 0.5 post-injury, minimal (score: 1) inflammatory infiltrate was present at the wound edge of WT mice (<i>upper right</i>), while significant (score: 3) inflammatory infiltrate was detected at the wound base. On the other hand, moderate (score: 2) and significant inflammatory infiltrates were observed at the wound edge (<i>lower right</i>) and base of FMOD-null mice, respectively. (<b>B</b>) At day 1 post-injury, moderate and significant inflammatory infiltrates were seen at the wound edge (<i>upper right</i>) and base of WT mice, respectively. Meanwhile, high (score: 4) inflammatory infiltrate was observed at both the wound edge (<i>lower-right</i>) and base of FMOD-null mice. (<b>C</b>) Relative inflammatory infiltration (median, 25–75% quartile, min, max) in 8 animals per genotype (2 randomly chosen wound edge fields and 2 randomly chosen wound bed fields per animal; N = 32) was semi-quantitatively evaluated by three blinded reviewers. Yellow arrowheads: representative inflammatory cells (not all inflammatory cells are indicated); yellow circles: randomly chosen fields for inflammatory infiltration evaluation. Bar =  100 µm. *, significant difference determined by the Mann-Whitney test.</p

<i>In vitro</i> migration assay of primary dermal fibroblasts derived from adult WT and FMOD-null mice skin.

<p>Cell migration was documented by photographs taken immediately after scraping, as well as 24(<b>A</b>). Migration was quantified by measuring the average wound gap between the wound edges before and after the treatment, and calculated as: Cell migration (%)  =  (Gap_0h-Gap_24h)/Gap_0h ×100% (<b>B</b>). 100 pM TGF-β3 was used to inhibit dermal fibroblast migration <i>in vitro</i>, while 10 µM TβRI-specific inhibitor SB-431542 was used to block TβRI-mediated signal transduction. Bar  = 200 µm. N = 6; *, <i>P</i><0.05. Red stars indicate the significance that resulted from FMOD-deficiency; green stars indicate the significance that resulted from TGF-β3 application; and blue stars indicate the significance that resulted from SA-431542 blockage of TβRI.</p

Relative Immunostaining Intensity of TGF-β Receptors in Wounded WT and FMOD-null Adult Mice.

<p>* WT, wild-type; FN, FMOD-null; N/A, not applicable; −, negligible staining (<5%); +, minimal staining (5%–25%); ++, moderate staining (25%–50%); +++, strong staining (>50%).</p>†<p>In general, unwounded control skin contained very few inflammatory cells. The chart reflects the intensity of intracellular TGF-β receptor staining and not the actually number of inflammatory cells present.</p>‡<p>Due to no hair follicle regeneration was detected in the adult skin wounds in this study, the chart reflects the influence of the wounds on adjacent hair follicles.</p

A brief summary of the major effects of FMOD-deficiency on adult mouse wound healing.

<p>A brief summary of the major effects of FMOD-deficiency on adult mouse wound healing.</p