Periosteum plays an indispensable role in bone repair and reconstruction. To recapitulate the remarkable regenerative capacity of periosteum, a biomimetic tissue-engineered periosteum (TEP) was constructed via layer-by-layer bottom-up strategy utilizing polycaprolactone (PCL), collagen, and nano-hydroxyapatite composite nanofiber sheets seeded with bone marrow stromal cells (BMSCs). When combined with a structural bone allograft to repair a 4 mm segmental bone defect created in the mouse femur, TEP restored donor-site periosteal bone formation, reversing the poor biomechanics of bone allograft healing at 6 weeks post-implantation. Further histologic analyses showed that TEP recapitulated the entire periosteal bone repair process, as evidenced by donor-dependent formation of bone and cartilage, induction of distinct CD31 type H endothelium, reconstitution of bone marrow and remodeling of bone allografts. Compared to nanofiber sheets without BMSC seeding, TEP eliminated the fibrotic tissue capsule elicited by nanofiber sheets, leading to a marked improvement of osseointegration at the compromised periosteal site. Taken together, our study demonstrated a novel layer-by-layer engineering platform for construction of a versatile biomimetic periosteum, enabling further assembly of a multi-component and multifunctional periosteum replacement for bone defect repair and reconstruction.
Objective. Connective tissue disease (CTD)-associated pulmonary arterial hypertension (PAH) is the second most common etiology of PAH and carries a poor prognosis. Recently, it has been shown that female human tumor necrosis factor (TNF)-transgenic (Tg) mice die of cardiopulmonary disease by 6 months of age. This study was undertaken to characterize this pathophysiology and assess its potential as a novel model of CTD-PAH. Methods. Histologic analysis was performed on TNF-Tg and wild-type (WT) mice to characterize pulmonary vascular and right ventricular (RV) pathology (n = 40 [4-5 mice per group per time point]). Mice underwent right-sided heart catheterization (n = 29) and micro-computed tomographic angiography (n = 8) to assess vascular disease. Bone marrow chimeric mice (n = 12), and anti-TNF-treated mice versus placebo-treated mice (n = 12), were assessed. RNA sequencing was performed on mouse lung tissue (n = 6). Results. TNF-Tg mice displayed a pulmonary vasculopathy marked by collagen deposition (P < 0.001) and vascular occlusion (P < 0.001) with associated RV hypertrophy (P < 0.001) and severely increased RV systolic pressure (mean ± SD 75.1 ± 19.3 mm Hg versus 26.7 ± 1.7 mm Hg in WT animals; P < 0.0001). TNF-Tg mice had increased α-smooth muscle actin (α-SMA) staining, which corresponded to proliferation and loss of von Willebrand factor (vWF)-positive endothelial cells (P < 0.01). There was an increase in α-SMA-positive, vWFpositive cells (P < 0.01), implicating endothelial-mesenchymal transition. Bone marrow chimera experiments revealed that mesenchymal but not bone marrow-derived cells are necessary to drive this process. Treatment with anti-TNF therapy halted the progression of disease. This pathology closely mimics human CTD-PAH, in which patient lungs demonstrate increased TNF signaling and significant similarities in genomic pathway dysregulation. Conclusion. The TNF-Tg mouse represents a novel model of CTD-PAH, recapitulates key disease features, and can serve as a valuable tool for discovery and assessment of therapeutics.
Thy-1 (CD90) is a well-known marker of fibroblasts implicated in organ fibrosis, but its contribution to skin fibrosis remains unknown. We examined Thy-1 expression in scleroderma skin and its potential role as a biomarker and pathogenic factor in animal models of skin fibrosis. Skin from patients with systemic sclerosis demonstrates markedly elevated Thy-1 expression compared to controls, co-localizes with fibroblast activator protein (FAP) in the deep dermis, and is correlated with the severity of skin involvement (MRSS). Serial imaging of skin from Thy-1 YFP reporter mice by IVIS showed an increase in Thy-1 expression which correlated with onset and progression of fibrosis. In contrast to lung fibrosis, Thy-1 KO mice had attenuated skin fibrosis in both bleomycin and Tsk-1 murine models. Moreover, Thy-1 regulated key pathogenic pathways involved in fibrosis including inflammation, myofibroblast differentiation, apoptosis and multiple additional canonical fibrotic pathways. Therefore, while Thy-1 deficiency leads to exacerbated lung fibrosis, in skin it is protective. Moreover, Thy-1 may serve as a longitudinal marker to assess skin fibrosis. Keywords: scleroderma, skin, fibrosis, Thy-1, fibroblasts expression (36.8 ± 11.1%) and later stage patients showing high expression (69.2 ± 8.9%) (figure 1A-C). It is known that FAP and Thy-1 expression status define subsets of fibroblasts in the skin with FAP + cells localizing primarily to the papillary dermis and Thy-1 + fibroblasts to the reticular dermis with a small number of double positive cells in the transition zone(13). Therefore, to further characterize SSc fibroblasts, we assessed co-expression of Thy-1 with FAP (Figure 1A, Supplemental figure 1 for higher magnification) and observed a remarkable change to FAP + Thy-1 + cells in SSc (45.3 ± 6.9% Thy-1 + cells double positive, compared to 1.4 ± 1.1% in controls) but did not note substantial differences in double positive cells in early vs late SSc. We next explored the gene expression of Thy-1 in skin from SSc patients in two publicly available microarray datasets (GSE58095 and GSE76886). We observed a significant increase in expression of Thy-1 in SSc across cohorts and elevated levels in both limited and diffuse cutaneous SSc (figure 1A, B, Supplemental figure 2). We also found a striking positive correlation between Thy-1 expression and severity of skin fibrosis measured by the modified Rodnan skin score (MRSS) (figure 1D, Spearman R = 0.63, p<0.0001). We next assessed Thy-1 lung expression in a publicly available data set (GSE48149) and found that Thy-1 was significantly elevated in SSc lung explants (Supplemental figure 3). This dataset included SSc patients with either ILD or pulmonary hypertension, patients with idiopathic pulmonary hypertension, and patients with idiopathic pulmonary fibrosis (IPF). Notably, all SSc patients and IPF patients had significantly elevated Thy-1 with a trend toward the largest increase in patients with SSc-ILD. A second smaller study (GSE81292) did not show any significant ...
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