ABSTRACT
The main structural component of connective tissues is fibrillar, cross-linked collagen whose fibrillogenesis can be modulated by Small Leucine-Rich Proteins/Proteoglycans (SLRPs). Not all SLRPs' effects on collagen and extracellular matrix in vivo have been elucidated; one of the less investigated SLRPs is asporin. Here we describe the successful generation of an Aspn-/- mouse model and the investigation of the Aspn-/- skin phenotype. Functionally, Aspn-/- mice had an increased skin mechanical toughness, although there were no structural changes present on histology or immunohistochemistry. Electron microscopy analyses showed 7% thinner collagen fibrils in Aspn-/- mice (not statistically significant). Several matrix genes were upregulated, including collagens (Col1a1, Col1a2, Col3a1), matrix metalloproteinases (Mmp2, Mmp3) and lysyl oxidases (Lox, Loxl2), while lysyl hydroxylase (Plod2) was downregulated. Intriguingly no differences were observed in collagen protein content or in collagen cross-linking-related lysine oxidation or hydroxylation. The glycosaminoglycan content and structure in Aspn-/- skin was profoundly altered: chondroitin/dermatan sulfate was more than doubled and had an altered composition, while heparan sulfate was halved and had a decreased sulfation. Also, decorin and biglycan were doubled in Aspn-/- skin. Overall, asporin deficiency changes skin glycosaminoglycan composition, and decorin and biglycan content, which may explain the changes in skin mechanical properties.
Subject(s)
Biglycan/genetics , Decorin/genetics , Extracellular Matrix Proteins/deficiency , Founder Effect , Gene Expression Regulation , Skin/metabolism , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Animals , Biglycan/metabolism , Chondroitin Sulfates/genetics , Chondroitin Sulfates/metabolism , Collagen Type I/genetics , Collagen Type I/metabolism , Collagen Type I, alpha 1 Chain , Collagen Type III/genetics , Collagen Type III/metabolism , Decorin/metabolism , Dermatan Sulfate/analogs & derivatives , Dermatan Sulfate/genetics , Dermatan Sulfate/metabolism , Extracellular Matrix/genetics , Extracellular Matrix/metabolism , Extracellular Matrix Proteins/genetics , Female , Heparitin Sulfate/genetics , Heparitin Sulfate/metabolism , Keratan Sulfate/genetics , Keratan Sulfate/metabolism , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Mice , Mice, Knockout , Phenotype , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Skin/ultrastructureABSTRACT
This work studies osteoinduction and bone conduction in polyvinyl alcohol-tetraethylorthosilicate-alginate-calcium oxide (PTAC) biocomposite cryogels along with the synergistic effect of electrical stimulation. In vitro osteoinduction of C2C12 myoblast towards osteogenic lineage is demonstrated through alkaline phosphatase assay, scanning electron microscopy and energy dispersive X-ray spectroscopy. These results were followed by in vivo implantation studies of PTAC biocomposite cryogel scaffolds in the bone conduction chamber model depicting bone formation after 24 days based on immunohistological staining for osteogenic markers, i.e., collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and bone sialoprotein (BSP). Further, osteogenic differentiation of murine mesenchymal stem cells was studied with and without electrical stimulation. The q-PCR analysis shows that the electrically stimulated cryogels exhibit ~ 6 folds higher collagen type I and ~ 10 folds higher osteopontin mRNA level, in comparison to the unstimulated cryogels. Thus, PTAC biocomposite cryogels present osteoinductive and osteoconductive properties during in vitro and in vivo studies and support osteogenic differentiation of mesenchymal stem cells under the influence of electrical stimulation.
Subject(s)
Cryogels/chemistry , Animals , Collagen/metabolism , Electric Stimulation , Integrin-Binding Sialoprotein/metabolism , Mice , Microscopy, Electron, Scanning , Osteocalcin/metabolism , Osteopontin/metabolism , Spectrometry, X-Ray EmissionABSTRACT
Long bone defects can be managed by the induced membrane technique together with autologous bone graft. However, graft harvest is associated with donor site morbidity. This study investigates if a tricalcium phosphate hydroxyapatite scaffold can be used alone or in combination with bone active drugs to improve healing. Sprague Dawley rats (n = 40) were randomized into four groups. (A) scaffold, (B) BMP-7, (C) BMP-7 + scaffold, and (D) BMP-7 + scaffold + systemic bisphosphonate at 2 weeks. Locked femoral nailing was followed by 6 mm segment removal and implantation of an epoxy spacer. At 4 weeks, the spacers were removed and the defects grafted. Eleven weeks later, the bones were explanted for evaluation with radiography, manual assessment, micro-CT, histology, and Fourier Transform Infrared spectroscopy (FTIR). Isolated scaffolds (A) did not heal any defects, whereas the other treatments led to healing in 7/10 (B), 10/10 (C), and 9/10 (D) rats. Group D had greater volume of highly mineralized bone (p < 0.01) and higher bone volume fraction (p < 0.01) compared to all other groups. A synthetic scaffold + BMP-7 combined with a bisphosphonate improved the callus properties in a rat femoral critical size defect, compared to both BMP-7 and scaffold alone or the two combined.
