Temporal and spatial modulation of chondrogenic foci in subchondral microdrill holes by chitosan-glycerol phosphate/blood implants
"Subchondral drilling initiates a cartilage repair response involving formation of chondrogenic foci in the subchondral compartment. The purpose of this study was to structurally characterize these sites of chondrogenesis and to investigate the effects of chitosan-glycerol phosphate (GP)/blood implants on their formation.
Thirty-two New Zealand White rabbits received bilateral cartilage defects bearing four subchondral drill holes. One knee per rabbit was treated by solidifying a chitosan-GP/blood implant over the defect. After 1-56 days of repair, chondrogenic foci [and collagen fiber orientation] were characterized. Glycosaminoglycan and collagen type II were present throughout the foci while the upper zone expressed collagen type I and the lower zone collagen type X. Large chondrogenic foci had a stratified structure with flatter cells closer to the articular surface, and round or hypertrophic chondrocytes deeper in the drill holes that showed signs of calcification after 3 weeks of repair in control defects. Markers for pre-hypertrophic chondrocytes (Patched) and for proliferation (Ki-67) were detected within foci. Some cells displayed a columnar arrangement where collagen was vertically oriented. For treated defects, chondrogenic foci appeared 1-3 weeks later, foci were nascent and mature rather than resorbing, and foci developed closer to the articular surface.
Chondrogenic foci bear some similarities to growth cartilage and can give rise to a repair tissue that has similar zonal stratification as articular cartilage."
"immature articular cartilage functions as an articular-epiphyseal growth cartilage"
"In the control drill holes, blood clot formation was followed by inflammatory and marrow-derived stromal cell migration, synthesis of granulation tissue and chondrogenic foci formation, starting at 2 weeks post-operative"
"Chondrogenic foci were growing above the tidemark in flanking cartilage to become cartilaginous repair tissues and most were being resorbed from below by the endochondral ossification process"
"chitosan-GP/blood implants increased recruitment of inflammatory cells and marrow-derived stromal cells during this period, and induced angiogenesis and bone remodeling under treated defects. Chitosan-GP/blood implants also delayed chondrogenic foci formation [and decreased cartilagenous foci absorption]"
Chondrocyte differentiation was observed near the bone thus stem cells may need to attach to bone to differentiate.
"Treatment with chitosan-GP/blood implants did not appear to significantly alter the structural organization of
chondrogenic foci, but rather changed their timing, maturation and position relative to the articular surface."<-whether the implants or control is better for height growth is unclear.
"The delay in chondroinduction in treated drill holes may be directly related to the previously reported increase in blood vessel density under defects treated with chitosan-GP/blood implants"<-so chitosan-GP implants may be used as a measure of the effect of blood vessel density on height growth.
"the rapid synthesis of connective tissue hinders tissue regeneration and that slowing the repair process could allow regeneration by progenitor cells rather than scar formation"
Implication of Neutrophils and Alternatively Activated Arginase-1+ Macrophages
"Microfracture and drilling elicit a cartilage repair whose quality depends on subchondral bone repair. Alternatively activated (AA) macrophages express arginase-1[Arginase 1 is upregulated by LSJL], release angiogenic factors, and could be potential mediators of trabecular bone repair.
Chitosan-glycerol phosphate (GP)/blood implants elicit arginase-1+ macrophages in vivo through neutrophil-dependent mechanisms and improve trabecular bone repair of drilled defects compared with drilling alone.
Bilateral trochlear cartilage defects were created in 15 rabbits, microdrilled, and treated or not with chitosan-GP/blood implant to analyze AA macrophages, CD-31+ blood vessels, bone, and cartilage repair after 1, 2, or 8 weeks. Neutrophil and macrophage chemotaxis to rabbit subcutaneous implants of autologous blood and chitosan-GP (+/-blood) was quantified at 1 or 7 days. In vitro, sera from human chitosan-GP/blood and whole blood clots cultured at 37 degrees C were analyzed by proteomics and neutrophil chemotaxis assays.
Chitosan-GP/blood clots and whole blood clots released a similar profile of chemotactic factors (PDGF-BB[PDGFC was upregulated by LSJL], IL-8/CXCL8, MCP-1/CCL2[upregulated 3.691 fold by LSJL], and no IL-1beta or IL-6), although chitosan clot sera attracted more neutrophils in vitro. Subcutaneous chitosan-GP (+/-blood) implants attracted more neutrophils (P < .001) and AA macrophages than whole blood clots in vivo. In repairing subchondral drill holes, chitosan-GP/blood implant attracted more AA macrophages at 1 and 2 weeks and more blood vessels at 2 weeks compared with drilled controls. Treatment elicited a more complete woven bone repair at 8 weeks than controls (P = .0011) with a more uniform, integrated collagen type II+ cartilage repair tissue.
AA macrophages may play a role in the regeneration of subchondral bone, and chitosan-GP can attract and transiently accumulate these cells in the repair tissue. The resulting improved subchondral repair could be advantageous toward enhancing integration of a restored chondral surface to the subchondral bone."
"‘‘alternatively’’ activated (AA) macrophages (induced by IL-4) express arginase-1 and release angiogenic factors such as TGF-b1 and insulin-like growth factor I (IGF-1) without releasing TNF-a."<-so increasing IL-4 levels may be a way to grow taller.
"Blood clots and chitosan particles attract neutrophils, and hybrid chitosan clots stimulate transient neutrophil influx, subchondral angiogenesis, and bone remodeling,"<-the only neutrophil related protein affected over threshold in LSJL is Ngp which is downregulated.
"IL-8 and MCP-1 were the dominant factors present in whole blood clot and chitosan-GP/blood clot serum, along with lower levels of G-CSF[CSF2ra was downregulated by LSJL], eotaxin, and EGF[expression of some EGF related proteins was altered in LSJL. Upregulated: Hbegf, Egfr, Edil-3]. in all cultured clot serum"
"LPS/blood clots, as expected, released high levels of proinflammatory mediators including TNF-a, IL-6, MIP-1a, MIP-1b, and IL-1b"
Growth plate chondrocytes inhibit neo-angiogenesis -- a possible mechanism for tumor control.
"Tumors seldom grow across the articular cartilage and physeal plate. It is believed that avascular cartilage may inhibit the neo-vascularization of tumor spread. The conditioned medium of growth plate chondrocytes resisted the migration of ECV304 by approximately 41% in invasion assay. Growth plate chondrocytes were shown by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to have an insignificant effect on the viability of human endothelial cells and fibroblasts. However, the proliferation of human endothelial cells was significantly inhibited by growth plate chondrocytes. The inhibitory activity was up to 35% and specific to endothelial cells. Inhibition of blood vessel formation was also demonstrated in the chick chorioallantoic membrane (CAM) assay. These findings demonstrated that growth plate chondrocytes secrete anti-angiogenesis factor(s) which specifically inhibit both the migration and proliferation of endothelial cells."
"direct extension of tumor across the old epiphyseal scar has been reported in all cases with closed growth plates"
"GPC CM disrupted, whereas SaOS-2 CM promoted blood vessels formation"
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