Thrombospondin (TSP) signals focal adhesion disassembly (the intermediate adhesive state) through interactions with cell surface calreticulin (CRT). TSP or a peptide (hep I) of the active site induces focal adhesion disassembly through binding to CRT, which activates phosphoinositide 3-kinase (PI3K) and extracellular signal–related kinase (ERK) through Gαi2 proteins. Because CRT is not a transmembrane protein, it is likely that CRT signals as part of a coreceptor complex. We now show that low density lipoprotein receptor–related protein (LRP) mediates focal adhesion disassembly initiated by TSP binding to CRT. LRP antagonists (antibodies, receptor-associated protein) block hep I/TSP-induced focal adhesion disassembly. LRP is necessary for TSP/hep I signaling because TSP/hep I is unable to stimulate focal adhesion disassembly or ERK or PI3K signaling in fibroblasts deficient in LRP. LRP is important in TSP–CRT signaling, as shown by the ability of hep I to stimulate association of Gαi2 with LRP. The isolated proteins LRP and CRT interact, and LRP and CRT are associated with hep I in molecular complexes extracted from cells. These data establish a mechanism of cell surface CRT signaling through its coreceptor, LRP, and suggest a novel function for LRP in regulating cell adhesion.

Bone wound healing after surgical drilling/cutting initially involves a typical inflammatory response with a leukocyte-rich cell infiltrate whose professional phagocytes (neutrophils and macrophages) clear the wound site of various bacterial (if present), particulate, and insoluble components arising from the original wounding event. As part of this process, in a surgical model of bone repair in rats, osteopontin (OPN) secreted by macrophages – with its known mineral-binding properties arising from abundant calcium-binding phosphorylations and overall net negative charge – binds to the newly exposed mineralized surfaces of particulate bone debris and the osseous wound margins created by the drilling, as shown by high-resolution immunogold labeling and transmission electron microscopy. For bone debris powder, OPN serves as an opsonin for clearance by macrophage phagocytosis, as demonstrated in vitro by phagocytosis assays using cultured J774.A1 murine macrophages and OPN-coated microbeads. Macrophage-secreted OPN binding to the bone wound margins contributes to cement line (plane) formation with subsequent OPN additions to the cement line coming from osteoblast lineage cells arriving at this site to effect bone repair upon further osteoblast differentiation, and extracellular matrix deposition and mineralization. Such interfacial OPN is thought to contribute to the cell adhesion, cell signaling, and matrix mineralization events required to effectively integrate the new bone into the preexisting bone at the margins of the drill site.

Bone extracellular matrix (ECM) is a 3D network, composed of collagen type I and a number of other macromolecules, including glycosaminoglycans (GAGs), which stimulate signaling pathways that regulate osteoblast growth and differentiation. To model the ECM of bone for tissue regenerative approaches, dense collagen/chitosan (Coll/CTS) hybrid hydrogels were developed using different proportions of CTS to mimic GAG components of the ECM. MC3T3-E1 mouse calvaria preosteoblasts were seeded within plastically compressed Coll/CTS hydrogels with solid content approaching that of native bone osteoid. Dense, cellular Coll/CTS hybrids were maintained for up to 8 weeks under either basal or osteogenic conditions. Higher CTS content significantly increased gel resistance to collagenase degradation. The incorporation of CTS to collagen gels decreased the apparent tensile modulus from 1.82 to 0.33 MPa. In contrast, the compressive modulus of Coll/CTS hybrids increased in direct proportion to CTS content exhibiting an increase from 23.50 to 55.25 kPa. CTS incorporation also led to an increase in scaffold resistance to cell-induced contraction. MC3T3-E1 viability, proliferation, and matrix remodeling capability (via matrix metalloproteinase expression) were maintained. Alkaline phosphatase activity was increased up to two-fold, and quantification of phosphate mineral deposition was significantly increased with CTS incorporation. Thus, dense Coll/CTS scaffolds provide osteoid-like models for the study of osteoblast differentiation and bone tissue engineering.

Extracellular matrix (ECM) consists of a complex mixture of macromolecules such as collagens, proteoglycans, glycoproteins, and elastic fibers. ECM is essential to preserving tissue architecture, signaling to cells, and regulating calcification in mineralized tissues. Osteoblasts in culture secrete and assemble an extensive ECM rich in type I collagen, and other noncollagenous proteins that can be mineralized. Three-dimensional matrix models can be used in vitro to most appropriately resemble the geometry and biochemistry of natural ECMs. In the present study, MC3T3-E1 mouse calvarial preosteoblasts were cultured within a dense three-dimensional collagenous ECM-like scaffold produced through the method of plastic compression. Plastic compression rapidly produces scaffolds of collagen density approaching native tissue levels with enhanced biomechanical properties while maintaining the viability of resident cells. The proliferation, morphology, and gene expression of seeded MC3T3s, as well as collagen production and matrix mineralization, were investigated for up to 7 weeks in culture. Soluble collagen secretion ranged in concentration from 5 to 30 microg/mL over a 24-h period, concomitant with a steady rate of collagen mRNA expression. Expression of osteogenic markers such as tissue-nonspecific alkaline phosphatase (Alpl), bone sialoprotein (Bsp), and osteopontin (Opn) examined by biochemical assay and reverse transcription-polymerase chain reaction demonstrated cell differentiation. Pericellular voids of ECM around cells, together with evidence of MMP13 mRNA expression, suggested matrix remodeling. Ultrastructural analyses, X-ray microanalysis, micro-computed tomography, as well as Fourier-transform infrared and imaging all confirmed the formation of a calcium-phosphate mineral phase within the fibrillar collagen matrix. In conclusion, preosteoblastic MC3T3 cells seeded within an ECM-like dense collagen scaffold secrete matrix proteins and induce scaffold mineralization in a manner potentially appropriate for bone tissue engineering uses.