Mesenchymal stem cells derived from adipose tissue have become a widely investigated cell source to use in tissue engineering applications. However, an optimal delivery scaffold for these cells is ...still needed. A rapidly gelling, injectable chitosan sponge was proposed in this study as a potential candidate for a suitable delivery scaffold. The results demonstrated the ability to encapsulate the stem cells at a 97.6% encapsulation efficiency and that the cells maintain their viability within the sponge. With the potential of using this scaffold for bone tissue engineering, ALP activity assay and fluorescent imaging for osteocalcin proved the ability to differentiate the encapsulated cells into the osteogenic lineage. Furthermore, co‐encapsulation of pyrophosphatase within the sponge was investigated as a method to overcome the inhibitory effects that the sponge degradation by‐products have on mineralization. Alizarin Red S staining demonstrated the beneficial effects of adding pyrophosphatase, where a significant increase in mineralization levels was achieved.
A rapidly gelling, biomimetic, purine cross‐linked chitosan sponge supported the attachment, survival, osteogenic differentiation, and mineralization of adipose‐derived mesenchymal stem cells (ASCs). The sponge demonstrated osteoinductive properties on the encapsulated ASCs, making it more appealing for bone tissue engineering applications.
Pediatric patients with Osteogenesis Imperfecta (OI), a heritable connective tissue disorder, frequently suffer from long bone deformations. Surgical correction often results in bone non-unions, ...necessitating revision surgery with autogenous bone grafting using bone-marrow-derived stem cells (BM-SC) to regenerate bone. BM-SC harvest is generally invasive and limited in supply; thus, adipose tissue's stromal vascular fraction (SVF) has been introduced as an alternative stem cell reservoir. To elucidate if OI patients' surgical site dissected adipose tissue could be used as autologous bone graft in future, we investigated whether the underlying genetic condition alters SVF's cell populations and in vitro differentiation capacity. After optimizing SVF isolation, we demonstrate successful isolation of SVF of pediatric OI patients and non-OI controls. The number of viable cells was comparable between OI and controls, with about 450,000 per gram tissue. Age, sex, type of OI, disease-causing collagen mutation, or anatomical site of harvest did not affect cell outcome. Further, SVF-containing cell populations were similar between OI and controls, and all isolated SVF's demonstrated chondrogenic, adipogenic, and osteogenic differentiation capacity in vitro. These results indicate that SVF from pediatric OI patients could be used as a source of stem cells for autologous stem cell therapy in OI.
Transglutaminase activity, arising potentially from transglutaminase 2 (TG2) and Factor XIIIA (FXIIIA), has been linked to osteoblast differentiation where it is required for type I collagen and ...fibronectin matrix deposition. In this study we have used an irreversible TG-inhibitor to 'block -and-track' enzyme(s) targeted during osteoblast differentiation. We show that the irreversible TG-inhibitor is highly potent in inhibiting osteoblast differentiation and mineralization and reduces secretion of both fibronectin and type I collagen and their release from the cell surface. Tracking of the dansyl probe by Western blotting and immunofluorescence microscopy demonstrated that the inhibitor targets plasma membrane-associated FXIIIA. TG2 appears not to contribute to crosslinking activity on the osteoblast surface. Inhibition of FXIIIA with NC9 resulted in defective secretory vesicle delivery to the plasma membrane which was attributable to a disorganized microtubule network and decreased microtubule association with the plasma membrane. NC9 inhibition of FXIIIA resulted in destabilization of microtubules as assessed by cellular Glu-tubulin levels. Furthermore, NC9 blocked modification of Glu-tubulin into 150 kDa high-molecular weight Glu-tubulin form which was specifically localized to the plasma membrane. FXIIIA enzyme and its crosslinking activity were colocalized with plasma membrane-associated tubulin, and thus, it appears that FXIIIA crosslinking activity is directed towards stabilizing the interaction of microtubules with the plasma membrane. Our work provides the first mechanistic cues as to how transglutaminase activity could affect protein secretion and matrix deposition in osteoblasts and suggests a novel function for plasma membrane FXIIIA in microtubule dynamics.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Transglutaminase (TG) enzymes and protein crosslinking have long been implicated in the formation of mineralized tissues. The aim of this study was to analyze the expression, activity and function of ...TGs in differentiating osteoblasts to gain further insight into the role of extracellular matrix protein crosslinking in bone formation. MC3T3-E1 (subclone 14) pre-osteoblast cultures were treated with ascorbic acid and β-glycerophosphate to induce cell differentiation and matrix mineralization. Expression of TG isoforms was analyzed by RT-PCR. TG activity was assessed during osteoblast differentiation by in vitro biochemical assays and by in situ labeling of live cell cultures. We demonstrate that MC3T3-E1/C14 osteoblasts express two TG isoforms—TG2 and FXIIIA. Abundant TG activity was observed during cell differentiation which increased significantly after thrombin treatment, a result confirming the presence of FXIIIA in the cultures. Ascorbic acid treatment, which stimulated collagen secretion and assembly, also stimulated externalization of TG activity, likely from FXIIIA which was externalized upon this treatment as analyzed by immunofluoresence microscopy. Inhibition of TG activity in the cultures by cystamine resulted in complete abrogation of mineralization, attributable to decreased matrix accumulation and an arrested state of osteoblast differentiation as measured by decreased levels of bone sialoprotein, osteocalcin and alkaline phosphatase. Additional functional studies and substrate characterization showed that TG activity was required for the formation of a fibronectin–collagen network during the early stages of matrix formation and assembly. This network, in turn, appeared to be essential for further matrix production and progression of the osteoblast differentiation program, and ultimately for mineralization.
Transglutaminases (TGs) are protein crosslinking enzymes involved in cell adhesion and signaling and matrix stabilization and maturation, in many cell types and tissues. We previously described that ...in addition to transglutaminase 2 (TG2), cultured MC3T3-E1 osteoblasts also express the plasma TG Factor XIIIA (FXIIIA). Here we report on the expression and localization of FXIIIA in bone in vivo and provide confirmatory in vitro data. Immuno-histochemistry and in situ hybridization demonstrated that FXIIIA is expressed by osteoblasts and osteocytes in long bones formed by endochondral ossification (femur) and flat bones formed primarily by intramembranous ossification (calvaria and mandible). FXIIIA immuno-reactivity was localized to osteoblasts, osteocytes, and the osteoid. RT-PCR analysis revealed FXIIIA expression by both primary osteoblasts and by the MC3T3-E1 osteoblast cell line. Western blot analysis of bone and MC3T3-E1 culture extracts demonstrated that FXIIIA is produced mainly as a small, 37-kDa form. Sequential RT-PCR analysis using overlapping PCR primers spanning the full FXIIIA gene showed that the entire FXIIIA gene is expressed, thus indicating that the 37-kDa FXIIIA is not a splice variant but a product of posttranslational proteolytic processing. Forskolin inhibition of osteoblast differentiation revealed that FXIIIA processing is regulated by the protein kinase A pathway.
Secreted protein, acidic, cysteine-rich (SPARC) is a glycoprotein that binds to collagen type I and other proteins in the extracellular matrix. Using whole-exome sequencing to identify the molecular ...defect in two unrelated girls with severe bone fragility and a clinical diagnosis of osteogenesis imperfecta type IV, we identified two homozygous variants in SPARC (GenBank: NM_003118.3; c.497G>A p.Arg166His in individual 1; c.787G>A p.Glu263Lys in individual 2). Published modeling and site-directed mutagenesis studies had previously shown that the residues substituted by these mutations form an intramolecular salt bridge in SPARC and are essential for the binding of SPARC to collagen type I. The amount of SPARC secreted by skin fibroblasts was reduced in individual 1 but appeared normal in individual 2. The migration of collagen type I alpha chains produced by these fibroblasts was mildly delayed on SDS-PAGE gel, suggesting some overmodification of collagen during triple helical formation. Pulse-chase experiments showed that collagen type I secretion was mildly delayed in skin fibroblasts from both individuals. Analysis of an iliac bone sample from individual 2 showed that trabecular bone was hypermineralized on the material level. In conclusion, these observations show that homozygous mutations in SPARC can give rise to severe bone fragility in humans.
Bone is one of the most dynamic tissues in the human body that can heal following injury without leaving a scar. However, in instances of extensive bone loss, this intrinsic capacity of bone to heal ...may not be sufficient and external intervention becomes necessary. Several techniques are available to address this problem, including autogenous bone grafts and allografts. However, all these techniques have their own limitations. An alternative method is the technique of distraction osteogenesis, where gradual and controlled distraction of two bony segments after osteotomy leads to induction of new bone formation. Although distraction osteogenesis usually gives satisfactory results, its major limitation is the prolonged duration of time required before the external fixator is removed, which may lead to numerous complications. Numerous methods to accelerate bone formation in the context of distraction osteogenesis have been reported. A viable alternative to autogenous bone grafts for a source of osteogenic cells is mesenchymal stem cells from bone marrow. However, there are certain problems with bone marrow aspirate. Hence, scientists have investigated other sources for mesenchymal stem cells, specifically adipose tissue, which has been shown to be an excellent source of mesenchymal stem cells. In this paper, the potential use of adipose stem cells to stimulate bone formation is discussed.
Osteogenesis imperfecta (OI) type VI is a recessively inherited form of OI that is caused by mutations in
SERPINF1
, the gene coding for pigment-epithelium derived factor (PEDF). Here, we report on ...two apparently unrelated children with OI type VI who had the same unusual homozygous variant in intron 6 of
SERPINF1
(c.787-10C>G). This variant created a novel splice site that led to the in-frame addition of three amino acids to PEDF (p.Lys262_Ile263insLeuSerGln). Western blotting showed that skin fibroblasts with this mutation produced PEDF but failed to secrete it. Both children were treated with intravenous bisphosphonates, but the treatment of Individual 1 was switched to subcutaneous injections of denosumab (dose 1 mg per kg body weight, repeated every 3 months). An iliac bone sample obtained after 5 denosumab injections (and 3 months after the last injection) showed no change in the increased osteoid parameters that are typical of OI type VI, but the number of osteoclasts in trabecular bone was markedly increased. This suggests that the effect of denosumab on osteoclast suppression is of shorter duration in children with OI type VI than what has previously been reported on adults with osteoporosis.
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