The extracellular matrix (ECM) is a complex of self assembled macromolecules. It is composed predominantly of collagens, non-collagenous glycoproteins, hyaluronan and proteoglycans. ECM is not only a scaffold for the cells; it serves also as a reservoir for growth factors and cytokines and modulates the cell activation status and turnover. ECM should be considered a dynamic network of molecules secreted by cells that in turn regulate cell behavior by modulating their proliferation and differentiation. The ECM provides structural strength to tissues, maintaining a complex architecture around the cells and the shape of organs. Various cell types secrete different matrix molecules and the nature and the amount of these molecules change during developmental age. Cartilage ECM is composed mainly of two components defining its mechano-physical properties: the collagenous network, responsible for the tensile strength of the cartilage matrix, and the proteoglycans (mainly aggrecan), responsible for the osmotic swelling and the elastic properties of the cartilage tissue. The conversion of cartilage into bone requires several processes that directly involve the different ECM components. Homeostasis of cartilage and bone is maintained by complex mechanisms controlling turnover and remodeling of ECM. In bone, as well as in cartilage, the ECM resident cells produce local factors, inflammatory mediators, and matrix-degrading enzymes. Turnover and degradation of normal and pathological matrices are dependent on the responses of the local cell to auto and paracrine anabolic and catabolic pathway.

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.

During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50–70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.

Lycolic acid (PGA)-hyaluronan scaffold with platelet-rich-plasma (PRP) in drilling. Questions/purposes We asked whether (1) PRP immersed in a cell-free PGA-hyaluronan scaffold improves patient-reported 1-year outcomes for the Knee injury and Osteoarthritis Score (KOOS), and (2) implantation of the scaffold in combination with bone marrow stimulation leads to the formation of hyaline-like cartilage repair tissue. Patients and Methods We reviewed 52 patients who had arthroscopic implantation of the PGA-hyaluronan scaffold immersed with PRP in articular cartilage defects of the knee pretreated with Pridie drilling. Patients were assessed by KOOS. At 9 months followup, histologic staining was performed in specimens obtained from five patients to assess the repair tissue quality. Results The KOOS subscores improved for pain (55 to 91), symptoms (57 to 88), activities of daily living (69 to 86), sports and recreation (36 to 70), and quality of life (38 to 73). The histologic evaluation showed a homogeneous hyaline-like cartilage repair tissue. Conclusions The cell-free PGA-hyaluronan scaffold combined with PRP leads to cartilage repair and improved patient-reported outcomes (KOOS) during 12 months of followup. Histologic sections showed morphologic features of hyaline-like repair tissue. Long-term followup is needed to determine if the cartilage repair tissue is durable. Level of Evidence Level IV, therapeutic study. See the Guidelines for Authors for a complete description of levels of evidence....

Platelet-rich plasma (PRP), a cocktail of platelet growth factors and bioactive proteins, has been proposed as a therapeutic agent to restore damaged articular cartilage. We report the biological effect of the platelet lysate (PL), a PRP derivative, on primary human articular chondrocytes cultured under both physiological and inflammatory conditions. When added to the culture medium, PL induced a strong mitogenic response in the chondrocytes. The in vitro expanded cell population maintained a chondrogenic redifferentiation potential as revealed by micromass culture in vitro and ectopic cartilage formation in vivo. Further, in chondrocytes cultured in the presence of the proinflammatory cytokine interleukin-1α (IL-1α), the PL induced a drastic enhancement of the synthesis of the cytokines IL-6 and IL-8 and of neutrophil-gelatinase associated lipocalin, a lipocalin expressed during chondrocyte differentiation and inflammation. These events were mediated by the p38 MAP kinase and NF-κB pathways. We observed that inflammatory stimuli activated phospo-MAP kinase-activated protein kinase 2, a direct target of p38. The proinflammatory effect of the PL was a transient phenomenon; after an initial upregulation, we observed significant reduction of the NF-κB activity together with the repression of the inflammatory enzyme cyclooxygenase-2. Moreover, the medium of chondrocytes cultured in the simultaneous presence of PL and IL-1α, showed a significant enhancement of the chemoattractant activity versus untreated chondrocytes. Our findings support the concept that the platelet products have a direct beneficial effect on articular chondrocytes and could drive in sequence a transient activation and the resolution of the inflammatory process, thus providing a rational for their use as therapeutic agents in cartilage inflammation and damage.