No abstract

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2 12 ‫؍‬ 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(L-latic acid) scaffold under the BI ؉ T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI ؉ T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.

Although autologous chondrocyte implantation has already been in clinical use, chondrocyte dedifferentiation is problematic during proliferation culture. We attempted a three-dimensional (3D) collagen gel culture under chondrocyte proliferation with repeated passaging to prevent the chondrocytes dedifferentiation. Human auricular chondrocytes were cultured in 3D or conventional monolayer conditions, which reached a 1000-fold increase in cell numbers at passages 3 and 4, respectively. During multiplication, the chondrocytes in 3D culture showed greater suppression of collagen type I (COL1) and preservation of collagen type II (COL2) than those in monolayer. Tissue-engineered cartilage made of 3D cells also abundantly accumulated COL2 or proteoglycan and possessed favorable mechanical properties. The advantage of 3D cells may result from the similarity of microenvironments in cell-to-matrix adhesion or cell-to-cell contacts with that of native cartilage. The up-regulation of integrins and down-regulation of cadherins in the 3D cells mimicked the expression pattern of native cartilage, rather than that of monolayer cells. The silencing of integrin beta1 and Ob-cadherin expression by small interfering ribonucleic acid in the cultured chondrocytes led to the promotion of dedifferentiation and redifferentiation, respectively, indicating that the 3D collagen gel culture provided sufficient cell preparation and reduced chondrocyte dedifferentiation, which is regarded as a feasible strategy in autologous chondrocyte implantation.

To overcome the weak points of the present cartilage regenerative medicine, we applied a porous scaffold for the production of tissue-engineered cartilage with a greater firmness and a 3D structure. We combined the porous scaffolds with atelocollagen to retain the cells within the porous body. We conducted canine autologous chondrocyte transplants using biodegradable poly-L-lactic acid (PLLA) or poly-DL-lactic-co-glycolic acid (PLGA) polymer scaffolds, and morphologically and biochemically evaluated the time course changes of the transplants. The histological findings showed that the tissue-engineered constructs using PLLA contained abundant cartilage 1, 2, and 6 months after transplantation. However, the PLGA constructs did not possess cartilage and could not maintain their shapes. Biochemical measurement of the proteoglycan and type II collagen also supported the superiority of PLLA. The biodegradation of PLGA progressed much faster than that of PLLA, and the PLGA had almost disappeared by 2 months. The degraded products of PLGA may evoke a more severe tissue reaction at this early stage of transplantation than PLLA. The PLLA scaffolds were suitable for cartilage tissue engineering under immunocompetent conditions, because of the retarded degradation properties and the decrease in the severe tissue reactions during the early stage of transplantation.

Construction of the interpenetrating polymer network (IPN) in hydrogels has received increased attention because it not only improves their mechanical properties but also mimics the extracellular matrix, which can be used as cell culturing scaffolds for tissue engineering. Usually, IPN gels are prepared using separated procedures, in which primal networks, followed by other networks, are formed by adding chemical reagents or subjecting to external stimuli. Herein, we designed a one-pot and in situ gelation system, which involved strategic selection of precursors for constructing IPN gels by simply mixing them. This design involved two types of gelation processes: RADA16 peptide self-assembling and covalent bond formation between chitosan (CH) and N-hydroxysuccinimide ester-terminated poly­(ethylene glycol) (NHS–PEG–NHS). The gelation kinetics suggested that RADA16 peptide networks were formed independently, followed by the formation of CH/PEG networks in the mixture containing the three components. Culturing chondrocytes in CH/PEG/RADA16 demonstrated that construction of the IPN structure promoted the embedded chondrocyte properties for the formation of the articular cartilage. Moreover, lower inflammation and higher protein production were observed in mice implanted with CH/PEG/RADA16-containing chondrocytes than in those with clinically used atelocollagen gel, appealing the feasibility of the proposed IPN hydrogel design for use as cell culturing scaffolds in tissue regeneration.

Tissue reactions against poly-L-lactic acid (PLLA) in engineered cartilage may influence the size or maturity of regenerative tissue. To understand the biological events in these reactions, we subcutaneously transplanted engineered constructs of PLLA scaffolds with or without human chondrocytes or atelocollagen in nude mice and evaluated neovascularization and macrophage activation, which can be assessed even in nude mice. Although not showing cartilage regeneration, PLLA alone demonstrated dense localization of macrophages and blood vessels, as well as a high level of interleukin-1 beta and tissue hemoglobin at 2 and 8 weeks. Otherwise, constructs with PLLA and chondrocytes with or without atelocollagen (PLLA/cell/gel or PLLA/cell) formed mature cartilage by 8 weeks, which was more prominent in PLLA/cell/gel. Although accumulation of macrophages and blood vessels in PLLA/cell/gel and PLLA/cell was comparable with that in PLLA at 2 weeks, that in PLLA/cell/gel markedly decreased by 8 weeks, with blood vessels and macrophages excluded into non-cartilage areas. Macrophage migration inhibitory factor could be involved in these suppressed tissue reactions, because it was expressed in chondrocytes of engineered cartilage. Intense tissue reactions inevitably occurred in biopolymers alone, but it is possible that maturation of engineered cartilage suppressed these reactions, which may contribute to circumventing deformity or malformation of engineered tissues.

To optimize the chondrocyte numbers obtained after collagenase digestion for cartilage tissue engineering, we examined the enzyme concentration and incubation time for collagenase digestion. The appropriate cell density in the chondrocyte primary culture was also verified. The collagenase digestion conditions that maximized the viable cell numbers were 24 h in 0.15% and 0.3% collagenase, 6 h in 0.6%, and 4 h in 1.2%, leading to ∼5 × 10⁵ cells from 0.05 g. When seeded at 10,000 cells/cm², all of these cells became almost confluent within 1 week. Cells digested in 0.3% for 24 h or 0.6% for 6 h and seeded at 3000 cells/cm² may also be acceptable, and similarly reached confluence within 1 week. Results regarding expression of the p53, tumor necrosis factor-α, and interleukin-1β genes, as well as apoptosis enzyme-linked immunosorbent assay results, show that excessive collagenase exposure may decrease chondrocyte viability or activity. We recommend a 24-h incubation in 0.3% collagenase or 6 h in 0.6% collagenase, and a cell-seeding density of 3000-10,000 cells/cm². These conditions maximize the harvest of isolated chondrocytes from a small amount of biopsied tissue and significantly aid in obtaining a large quantity of cultured cells in a short period.

For improving the quality of tissue-engineered cartilage, we examined the in vivo usefulness of porous bodies as scaffolds combined with an atelocollagen hydrogel, and investigated the suitable conditions for atelocollagen and seeding cells within the engineered tissues. We made tissue-engineered constructs using a collagen sponge (CS) or porous poly(L-lactide) (PLLA) with human chondrocytes and 1% hydrogel, the concentration of which maximized the accumulation of cartilage matrices. The CS was soft with a Young's modulus of less than 1 MPa, whereas the porous PLLA was very rigid with a Young's modulus of 10 MPa. Although the constructs with the CS shrank to 50% in size after a 2-month subcutaneous transplantation in nude mice, the PLLA constructs maintained their original sizes. Both of the porous scaffolds contained some cartilage regeneration in the presence of the chondrocytes and hydrogel, but the PLLA counterpart significantly accumulated abundant matrices in vivo. Regarding the conditions of the chondrocytes, the cartilage regeneration was improved in inverse proportion to the passage numbers among passages 3-8, and was linear with the cell densities (10(6) to 10(8) cells/mL). Thus, the rigid porous scaffold can maintain the size of the tissue-engineered cartilage and realize fair cartilage regeneration in vivo when combined with 1% atelocollagen and some conditioned chondrocytes.