Solidification mechanisms of chitosan–glycerol phosphate/blood implant for articular cartilage repair

SummaryObjectiveChitosan–glycerol phosphate (chitosan-GP) is a unique polymer solution that is mixed with whole blood and solidified over microfractured or drilled articular cartilage defects in order to elicit a more hyaline repair cartilage. For clinical ease-of-use, a faster in situ solidification is preferred. Therefore, we investigated the mechanisms underlying chitosan–GP/blood implant solidification.MethodsIn vitro solidification of chitosan–GP/blood mixtures, with or without added clotting factors, was evaluated by thromboelastography. Serum was analyzed for the onset of thrombin, platelet, and FXIII activation. In vivo solidification of chitosan–GP/blood mixtures, with and without clotting factors, was evaluated in microdrilled cartilage defects of adult rabbits (N=41 defects).ResultsChitosan–GP/blood clots solidified in an atypical biphasic manner, with higher initial viscosity and minor platelet activation followed by the development of clot tensile strength concomitant with thrombin generation, burst platelet and FXIII activation. Whole blood and chitosan–GP/blood clots developed a similar final clot tensile strength, while polymer–blood clots showed a unique, sustained platelet factor release and greater resistance to lysis by tissue plasminogen activator. Thrombin, tissue factor (TF), and recombinant human activated factor VII (rhFVIIa) accelerated chitosan–GP/blood solidification in vitro (P<0.05). Pre-application of thrombin or rhFVIIa+TF to the surface of drilled cartilage defects accelerated implant solidification in vivo (P<0.05).ConclusionsChitosan–GP/blood implants solidify through coagulation mechanisms involving thrombin generation, platelet activation and fibrin polymerization, leading to a dual fibrin–polysaccharide clot scaffold that resists lysis and is physically more stable than normal blood clots. Clotting factors have the potential to enhance the practical use, the residency, and therapeutic activity of polymer–blood implants

Subchondral chitosan/blood implant-guided bone plate resorption and woven bone repair is coupled to hyaline cartilage regeneration from microdrill holes in aged rabbit knees

SummaryObjectiveLittle is known of how to routinely elicit hyaline cartilage repair tissue in middle-aged patients. We tested the hypothesis that in skeletally aged rabbit knees, microdrill holes can be stimulated to remodel the bone plate and induce a more integrated, voluminous and hyaline cartilage repair tissue when treated by subchondral chitosan/blood implants.DesignNew Zealand White rabbits (13 or 32 months old, N = 7) received two 1.5 mm diameter, 2 mm depth drill holes in each knee, either left to bleed as surgical controls or press-fit with a 10 kDa (distal hole: 10K) or 40 kDa (proximal hole: 40K) chitosan/blood implant with fluorescent chitosan tracer. Post-operative knee effusion was documented. Repair tissues at day 0 (N = 1) and day 70 post-surgery (N = 6) were analyzed by micro-computed tomography, and by histological scoring and histomorphometry (SafO, Col-2, and Col-1) at day 70.ResultsAll chitosan implants were completely cleared after 70 days, without increasing transient post-operative knee effusion compared to controls. Proximal control holes had worse osteochondral repair than distal holes. Both implant formulations induced bone remodeling and improved lateral integration of the bone plate at the hole edge. The 40K implant inhibited further bone repair inside 50% of the proximal holes, while the 10K implant specifically induced a “wound bloom” reaction, characterized by decreased bone plate density in a limited zone beyond the initial hole edge, and increased woven bone (WB) plate repair inside the initial hole (P = 0.016), which was accompanied by a more voluminous and hyaline cartilage repair (P < 0.05 vs control defects).ConclusionIn a challenging aged rabbit model, bone marrow-derived hyaline cartilage repair can be promoted by treating acute drill holes with a biodegradable subchondral implant that elicits bone plate resorption followed by anabolic WB repair within a 70-day repair period

Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure

Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10 · 106 cells/ mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4MPa Pulsatile HP; (2) 0.4MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10x106 cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4MPa Pulsatile HP; (2) 5MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.Fundação para a Ciência e a Tecnologia (FCT) - SFRH/BD/42316/200