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

Chitosan–glycerol phosphate/blood implants increase cell recruitment, transient vascularization and subchondral bone remodeling in drilled cartilage defects

SummaryObjectiveMarrow-stimulation techniques are used by surgeons to repair cartilage lesions although consistent regeneration of hyaline cartilage is rare. We have shown previously that autologous blood can be mixed with a polymer solution containing chitosan in a glycerol phosphate (GP) buffer (chitosan–GP), and that implantation of this polymer/blood composite onto marrow-stimulated chondral defects in rabbit and sheep leads to the synthesis of more chondral repair tissue with greater hyaline character compared to marrow-stimulation alone. In the current study, we examined the modulation of cell recruitment and repair tissue characteristics at early post-surgical time points (from day 1 to 56) in a rabbit model to elucidate potential mechanisms behind this improved repair outcome.DesignThirty-three skeletally mature New Zealand White rabbits underwent bilateral arthrotomies, with each trochlea receiving a cartilage defect (3.5mm×4.5mm) bearing four microdrill holes (0.9mm diameter, ∼4mm deep) into the subchondral bone. One defect per rabbit was treated with a chitosan–GP/blood implant, while the other defect was left as a microdrilled control. Repair tissues were stained by histochemistry, for collagen types I, II, and X by immunohistochemistry and analyzed using quantitative stereological tools.ResultsHistological analyses demonstrated that control defects followed a typical healing sequence observed previously in marrow-stimulation animal models while chitosan–GP/blood implants led to three significant modifications in the healing sequence at early stages: (1) increased inflammatory and marrow-derived stromal cell recruitment to the microdrill holes, (2) increased vascularization of the provisional repair tissue in the microdrill holes, and (3) increased intramembranous bone formation and subchondral bone remodeling (BR).ConclusionsThese results suggest that the greater levels of provisional tissue vascularization and BR activity are main factors supporting improved cartilage repair when chitosan–GP/blood implants are applied to marrow-stimulated cartilage lesions

Temporal and spatial modulation of chondrogenic foci in subchondral microdrill holes by chitosan-glycerol phosphate/blood implants

SummaryObjectiveSubchondral 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.MethodThirty-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 were characterized by histostaining and immunostaining. Collagen fiber orientation was characterized by polarized light microscopy.ResultsGlycosaminoglycan 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.ConclusionsChondrogenic foci bear some similarities to growth cartilage and can give rise to a repair tissue that has similar zonal stratification as articular cartilage. The temporal and spatial formation of chondrogenic foci can be modulated by cartilage repair therapies

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

Tissue engineering of cartilage using an injectable and adhesive chitosan-based cell-delivery vehicle

SummaryObjectiveAdult articular cartilage shows a limited intrinsic repair response to traumatic injury. To regenerate damaged cartilage, cell-assisted repair is thus viewed as a promising therapy, despite being limited by the lack of a suitable technique to deliver and retain chondrogenic cells at the defect site.DesignWe have developed a cytocompatible chitosan solution that is space-filling, gels within minutes, and adheres to cartilage and bone in situ. This unique combination of properties suggested significant potential for its use as an arthroscopically injectable vehicle for cell-assisted cartilage repair. The primary goal of this study was to assess the ability of this polymer system, when loaded with primary articular chondrocytes, to support cartilage formation in vitro and in vivo. The chitosan gel was cultured in vitro, with and without chondrocytes, as well as injected subcutaneously in nude mice to form subcutaneous dorsal implants. In vitro and in vivo constructs were collectively analyzed histologically, for chondrocyte mRNA and protein expression, for biochemical levels of glycosaminoglycan, collagen, and DNA, and for mechanical properties.ResultsResulting tissue constructs revealed histochemical, biochemical and mechanical properties comparable to those observed in vitro for primary chondrocytes cultured in 2% agarose. Moreover, the gel was retained after injection into a surgically prepared, rabbit full-thickness chondral defect after 1 day in vivo, and in rabbit osteochondral defects, up to 1 week.ConclusionsThe in situ-gelling chitosan solution described here can support in vitro and in vivo accumulation of cartilage matrix by primary chondrocytes, while persisting in osteochondral defects at least 1 week in vivo