Hydrolysis and Dissolution of Amyloids by Catabodies.

Catalytic antibodies (catabodies) hold potential for superior immunotherapy because of their turnover capability and no or minimal induction of inflammatory responses. Catabodies neutralize and remove target antigens more potently than conventional antibodies. Depending on the catalytic rate constant, a single catabody molecule degrades thousands to millions of target molecules over its useful lifespan, whereas conventional antibodies only form reversibly associated, stoichiometric complexes with the target. Thus, removal of the antibody-bound target requires accessory phagocytic cells that ingest the immune complexes, which is usually accompanied by release of inflammatory mediators. In comparison, catabodies bind the target only transiently, and the rapid and direct target destruction reduces the concentration of immune complexes that can activate inflammatory processes. These features are especially pertinent when large target amounts at anatomically vulnerable sites must be removed, e.g., amyloids. We reported specific catabodies to misfolded transthyretin (misTTR) amyloid and amyloid ß peptide (Aß). Accumulation of the oligomeric and fibrillized amyloid TTR forms causes diverse systemic pathologies, including cardiomyopathy, polyneuropathy, and skeletal diseases. Brain Aß aggregates are thought to cause central nervous system degenerative disease, chiefly Alzheimer's disease. We describe methods for testing catabody-mediated degradation and dissolution of Aß and TTR.

Enhanced osteogenicity of bioactive composites with biomimetic treatment.

PURPOSE: This study aimed to explore if initiation of biomimetic apatite nucleation can be used to enhance osteoblast response to biodegradable tissue regeneration composite membranes. MATERIALS AND METHODS: Bioactive thermoplastic composites consisting of poly(ε-caprolactone/DL-lactide) and bioactive glass (BAG) were prepared at different stages of biomimetic calcium phosphate deposition by immersion in simulated body fluid (SBF). The modulation of the BAG dissolution and the osteogenic response of rat mesenchymal stem cells (MSCs) were analyzed. RESULTS: SBF treatment resulted in a gradual calcium phosphate deposition on the composites and decreased BAG reactivity in the subsequent cell cultures. Untreated composites and composites covered by thick calcium phosphate layer (14 days in SBF) expedited MSC mineralization in comparison to neat polymers without BAG, whereas other osteogenic markers--alkaline phosphatase activity, bone sialoprotein, and osteocalcin expression--were initially decreased. In contrast, surfaces with only small calcium phosphate aggregates (five days in SBF) had similar early response than neat polymers but still demonstrated enhanced mineralization. CONCLUSION: A short biomimetic treatment enhances osteoblast response to bioactive composite membranes.

Enhancing chondrogenic phenotype for cartilage tissue engineering: monoculture and coculture of articular chondrocytes and mesenchymal stem cells.

Articular cartilage exhibits an inherently low rate of regeneration. Consequently, damage to articular cartilage often requires surgical intervention. However, existing treatments generally result in the formation of fibrocartilage tissue, which is inferior to native articular cartilage. As a result, cartilage engineering strategies seek to repair or replace damaged cartilage with an engineered tissue that restores full functionality to the impaired joint. These strategies often involve the use of chondrocytes, yet in vitro expansion and culture can lead to undesirable changes in chondrocyte phenotype. This review focuses on the use of articular chondrocytes and mesenchymal stem cells (MSCs) in either monoculture or coculture for the enhancement of chondrogenesis. Coculture strategies increasingly outperform their monoculture counterparts with regard to chondrogenesis and present unique opportunities to attain chondrocyte phenotype stability in vitro. Methods to prevent chondrocyte dedifferentiation and promote chondrocyte redifferentiation as well as to promote the chondrogenic differentiation of MSCs while preventing MSC hypertrophy are discussed.

Chondrogenic phenotype of articular chondrocytes in monoculture and co-culture with mesenchymal stem cells in flow perfusion.

This work investigated the effect of flow perfusion bioreactor culture with and without transforming growth factor-ß3 (TGF-ß3) supplementation on the proliferation, extracellular matrix (ECM) production, and chondrogenic gene expression of chondrocytes both in monoculture and in co-culture with bone marrow-derived mesenchymal stem cells (MSCs). Both cell populations were cultured on electrospun poly(ɛ-caprolactone) scaffolds for 2 weeks in static or flow perfusion culture with and without TGF-ß3. Overall, it was observed that without growth factors, flow perfusion culture resulted in increased cell proliferation and ECM with a more cartilage-like composition. While with TGF-ß3 induction, flow perfusion constructs generally had lower chondrogenic gene expression than the corresponding static cultures, the growth factor still had an inductive effect on the cells with enhanced gene expression compared with the corresponding noninduced cultures. In addition, while flow perfusion cultures generally had reduced overall ECM content, the ECM distribution was more homogenous compared with the corresponding static cultures. These results are significant in that they indicate that while flow perfusion culture has some beneficial effects on the chondrogenic phenotype of articular chondrocytes, flow perfusion alone is not sufficient to maintain the chondrogenic phenotype of chondrocytes in either monoculture or co-culture, thus demonstrating the advantages of using exogenously added growth factors in flow perfusion culture. Furthermore, the results demonstrate the advantages of flow perfusion culture for the creation of large tissue engineered constructs and the potential of co-cultures of articular chondrocytes and MSCs to be used in flow perfusion culture.

Articular chondrocyte redifferentiation in 3D co-cultures with mesenchymal stem cells.

In this work, we evaluated the ability of 3D co-cultures with mesenchymal stem cells (MSCs) to redifferentiate monolayer expanded articular chondrocytes (ACs) and produce cartilaginous extracellular matrix at varying stages of the dedifferentiation process and further examined the dependency of this effect on the culture medium composition. Primary bovine ACs were expanded in monolayers for up to nine population doublings to obtain seven cell stocks with gradually increasing levels of dedifferentiation. Culture expanded ACs were then seeded as monocultures and co-cultures with rabbit bone marrow-derived MSCs (30:70 ratio of ACs-to-MSCs) on porous scaffolds. Parallel cultures were established for each cell population in serum-containing growth medium and serum-free induction medium supplemented with dexamethasone and TGF-ß3. After 3 weeks, all groups were analyzed for DNA content, glycosaminoglycan (GAG) and hydroxyproline (HYP) production, and chondrogenic gene expression. Significant enhancements in cellularity, GAG content and GAG/HYP ratio, and chondrogenic phenotype were observed in the induction medium compared to growth medium at all levels of AC expansion. Furthermore, primary co-cultures showed similarly enhanced chondrogenesis compared to monocultures in both culture media, whereas passaged ACs benefitted from co-culturing only in the induction medium. We conclude that co-cultures of ACs and MSCs can produce superior in vitro engineered cartilage in comparison to pure AC cultures, due to both heterotypic cellular interactions and decreased need for monolayer expansion of biopsied chondrocytes. While the initial level of AC dedifferentiation affected the quality of the engineered constructs, co-culture benefits were realized at all stages of AC expansion when suitable chondroinductive culture medium was used.

Ectopic bone formation in and soft-tissue response to P(CL/DLLA)/bioactive glass composite scaffolds.

OBJECTIVES: To characterize biological response to subcutaneously implanted macroporous poly(ε-caprolactone/D,L-lactide)-based scaffolds, and to evaluate the effect of bioactive glass (BAG) filler and osteogenic cells to the tissue response and ectopic bone formation. MATERIAL AND METHODS: In the first part of this study, six different scaffold types were screened in a rat subcutaneous implantation model. The polymer scaffolds with 70/30 caprolactone/lactide ratio and corresponding composites with < 45 µm BAG filler size were chosen for the further ectopic bone formation assay. The scaffolds were loaded with differentiating bone marrow stromal cells and implanted subcutaneously in syngeneic rats. RESULTS: With plain scaffolds, only mild foreign body reaction with no signs of gross inflammation was observed after 4 weeks of implantation. Furthermore, the scaffolds were fully invaded by well-vascularized soft connective tissue. Overall, all the tested scaffold types showed an appropriate host response. With cell-seeded scaffolds, several loci of immature mineralizing tissue and small amounts of mature bone were observed after 4 weeks. The incidence of mature bone formation was two and four in polymer scaffolds and composites, respectively (n = 8). After twelve weeks, mature bone was observed in only one polymer scaffold but in seven composites (n = 8). Excluding bone formation, the host response was considered similar to that with cell-free scaffolds. CONCLUSIONS: Plain scaffolds supported the ingrowth of well-vascularized fibroconnective tissue. Furthermore, cell seeded composites with BAG filler showed enhanced ectopic bone formation in comparison with corresponding neat polymer scaffolds.

TGF-ß3-induced chondrogenesis in co-cultures of chondrocytes and mesenchymal stem cells on biodegradable scaffolds.

In this work, it was hypothesized that co-cultures of articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) would exhibit enhanced sensitivity to chondrogenic stimuli, such as TGF-ß3, and would require a reduced concentration of TGF-ß3 to achieve an equivalent level of chondrogenesis compared to monocultures of each cell type. Furthermore, it was hypothesized that compared to monocultures, the chondrogenic phenotype of AC/MSC co-cultures would be more stable upon the removal of TGF-ß3 from the culture medium. These hypotheses were investigated by culturing ACs and MSCs alone and in a 1:3 ratio on electrospun poly(ε-caprolactone) scaffolds. All cell populations were cultured for two weeks with 0, 1, 3, or 10 ng/ml of TGF-ß3. After two weeks growth factor supplementation was removed, and the constructs were cultured for two additional weeks. Cell proliferation, extracellular matrix production, and chondrogenic gene expression were evaluated after two and four weeks. The results demonstrated that co-cultures of ACs and MSCs require a reduced concentration and duration of TGF-ß3 exposure to achieve an equivalent level of chondrogenesis compared to AC or MSC monocultures. Thus, the present work implicates that the promise of co-cultures for cartilage engineering is enhanced by their robust phenotype and heightened sensitivity to TGF-ß3.

Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels.

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-ß3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7 days, but not 14 days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28 days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.

The effect of hypoxia on the chondrogenic differentiation of co-cultured articular chondrocytes and mesenchymal stem cells in scaffolds.

In this work, we investigated the effects of lowered oxygen tension (20% and 5% O2) on the chondrogenesis and hypertrophy of articular chondrocytes (ACs), mesenchymal stem cells (MSCs) and their co-cultures with a 30:70 AC:MSC ratio. Cells were cultured for six weeks within porous scaffolds, and their cellularity, cartilaginous matrix production (collagen II/I expression ratio, hydroxyproline and GAG content) and hypertrophy markers (collagen X expression, ALP activity, calcium accumulation) were analyzed. After two weeks, hypoxic culture conditions had expedited chondrogenesis with all cell types by increasing collagen II/I expression ratio and matrix synthesis by ~2.5-11 and ~1.5-3.0 fold, respectively. At later times, hypoxia decreased cellularity but had little effect on matrix synthesis. ACs and co-cultures showed similarly high collagen II/I expression ratio and GAG rich matrix formation, whereas MSCs produced the least hyaline cartilage-like matrix and obtained a hypertrophic phenotype with eventual calcification. MSC hypertrophy was further emphasized in hypoxic conditions. We conclude that the most promising cell source for cartilage engineering was co-cultures, as they have a potential to decrease the need for primary chondrocyte harvest and expansion while obtaining a stable highly chondrogenic phenotype independent of the oxygen tension in the cultures.

Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells.

In this work, articular chondrocytes (ACs) and mesenchymal stem cells (MSCs) with 1:1 and 1:3 cell ratios were co-cultured in order to evaluate if a majority of primary ACs can be replaced with MSCs without detrimental effects on in vitro chondrogenesis. We further used a xenogeneic culture model to study if such co-cultures can result in redifferentiation of passaged ACs. Cells were cultured in porous scaffolds for four weeks and their cellularity, cartilage-like matrix formation and chondrogenic gene expression levels (collagen I and II, aggrecan) were measured. Constructs with primary bovine ACs had ~1.6 and 5.5 times higher final DNA and glycosaminoglycan contents, respectively, in comparison to those with culture expanded chondrocytes or MSCs harvested from the same animals. Equally robust chondrogenesis was also observed in co-cultures, even when up to 75% of primary ACs were initially replaced with MSCs. Furthermore, species-specific RT-PCR analysis indicated a gradual loss of MSCs in bovine-rabbit co-cultures. Finally, co-cultures using primary and culture expanded ACs resulted in similar outcomes. We conclude that the most promising cell source for cartilage engineering was the co-cultures, as the trophic effect of MSCs may highly increase the chondrogenic potential of ACs thus diminishing the problems with primary chondrocyte harvest and expansion.

Design of a high-throughput flow perfusion bioreactor system for tissue engineering.

Flow perfusion culture is used in many areas of tissue engineering and offers several key advantages. However, one challenge to these cultures is the relatively low-throughput nature of perfusion bioreactors. Here, a flow perfusion bioreactor with increased throughput was designed and built for tissue engineering. This design uses an integrated medium reservoir and flow chamber in order to increase the throughput, limit the volume of medium required to operate the system, and simplify the assembly and operation.

Photo-cross-linked biodegradable poly(ester anhydride) networks prepared from alkenylsuccinic anhydride functionalized poly(ε-caprolactone) precursors.

Biodegradable poly(ester anhydride) networks based on linear and star-shaped poly(ε-caprolactone)-based precursors were synthesized with the aim of obtaining matrixes suitable for release of macromolecular active agents. The ring-opening polymerization yielded hydroxyl telechelic oligomers, which were end-functionalized with succinic anhydride or with alkenylsuccinic anhydrides containing 8, 12, or 18 carbons in their alkenyl chains. Before cross-linking, the acid-terminated oligomers were reacted with methacrylic anhydride to obtain methacrylated precursors containing labile anhydride bonds. The degrees of substitution for the acid functionalization and methacrylation were >93%. Cross-linking of the precursors was carried out with visible light at room temperature. Gel contents and cross-linking densities were higher for networks cross-linked from the star-shaped precursors than for networks prepared from the linear precursors. In in vitro erosion tests, the presence of the alkenyl chain slowed down the erosion rate. The networks exhibited characteristic surface erosion: the mass loss was linear, whereas the dimensions of the specimens decreased steadily. A macromolecular release study showed the release of the model compound to be linear and in proportion to the mass loss.

Osteoblast response to continuous phase macroporous scaffolds under static and dynamic culture conditions.

Average scaffold pore sizes in the order of several hundred microns are generally required for efficient bone tissue ingrowth in vivo, whereas the culture of large bone engineering constructs in vitro can require bioreactor cultures to decrease diffusional constraints on the cells. In this study, we prepared poly(epsilon-caprolactone/D,L-lactide)-based scaffolds with continuous phase macroporosity using a novel CaCl(2) . 6H(2)O porogen agent. Osteogenic differentiation and scaffold colonization in rat bone marrow stromal cell cultures were compared in such polymer scaffolds, and in composites with 30 wt % bioactive glass filler. The effect of a rotating wall bioreactor culture on the cell response was also evaluated. Bioactive filler enhanced proliferation, early osteogenic differentiation, and mineralization of the cultured cells under static conditions. Dynamic cultures, in turn, resulted in decreased cell numbers and inhibition of the differentiation process irrespective of the scaffold type. This effect was ascribed to the harsh mechanical stresses caused by constant collisions of the scaffolds in the bioreactor vessels. However, cells were able to penetrate into the scaffold interior only under dynamic culture conditions. Thus, interconnected macroporosity is an essential, but not sufficient, condition to allow for full colonization of millimeter scale tissue engineering scaffolds in vitro.

Osteoblast proliferation and maturation on bioactive fiber-reinforced composite surface.

The objective of this study was to evaluate the proliferation and osteogenic potential of bone-marrow derived osteoblast-like cells on fiber-reinforced composite (FRC) substrates with and without bioactive glass surface modification. Three FRC materials were fabricated for the study: (a) grit-blasted FRC, (b) grit-blasted FRC with bidirectional net reinforcement and (c) FRC with bioactive glass (BAG) coating. Rat bone-marrow derived osteoblast-like cells were harvested and cultured on experimental material plates and on cp. titanium plates (control) for 21 days. The materials' surfaces were characterized by roughness testing and scanning electron microscopy. Cell growth and differentiation kinetics were subsequently investigated by evaluating proliferation, alkaline phosphatase (ALP) activity, osteocalcin (OC) and bone sialoprotein (BSP) production. On day 14, the cell proliferation was significantly lower (P<0.05) on FRC-BAG than on titanium and FRC. The proliferation on the other three materials was equal throughout the experiment. The maximal ALP activities on FRC, FRC-Net, and titanium were observed on day 21, whereas FRC-BAG had already reached the maximal level on day 14. Expression of osteoblastic markers (OC, BSP) indicates that the fastest osteogenic differentiation takes place on FRC after 7 days. In contrast, a slower differentiation process was observed on titanium than on any other tested material (P<0.015) at 21 days, as was confirmed by increased mRNA expression of OC and BSP. It can be concluded that the proliferation and maturation of osteoblast-like cells on FRC appears to be comparable to titanium. Presence of BAG enhances cell maturation.

Titania and titania-silica coatings for titanium: comparison of ectopic bone formation within cell-seeded scaffolds.

The aim of this study was to compare titania (TiO(2))-coated, titania-silica (TiSi)-coated, and uncoated (cpTi) titanium fiber meshes as scaffolds for bone engineering. The scaffolds were loaded with bone marrow stromal cells and implanted subcutaneously in rats. Ectopic bone formation after 1, 4, and 12 weeks of implantation was evaluated using histology and histomorphometry. After 1 week of implantation, multiple patches of unorganized mineralizing tissue were seen in all implants. The amount of this bone-like tissue clearly increased from 1 to 4 weeks. Bone apposition occurred in direct contact with coated meshes, while a thin layer of unmineralized fibrous tissue was often observed surrounding cpTi mesh fibers. After 12 weeks, the structure of bone, with bone marrow-like tissue, was further matured and mesh fibers were embedded in lamellar bone. No statistical differences in the amount of mineralized bone were observed between scaffold types at any point of time. Only TiSi scaffolds showed further increase in bone area from 4 to 12 weeks (p < 0.01). A notable difference was that the sol-gel coatings resulted in enhanced initial bone contact and distribution of bone tissue, whereas uncoated implants showed bone formation mainly in the center of the scaffolds. In conclusion, TiO(2)-based sol-gel coatings may be used in tissue engineering to gain more uniform distribution of bone throughout titanium fiber mesh scaffolds.

Osteoblast differentiation with titania and titania-silica-coated titanium fiber meshes.

Two surface-reactive sol-gel coatings, namely titania (TiO2) and a mixture of titania and silica (TiSi), were applied to titanium fiber meshes. Differentiation of rat bone marrow stromal cells toward an osteogenic phenotype with coated and uncoated (cpTi) substrates was compared. The amount of DNA in cpTi and TiSi matrices did not increase after day 3, but with TiO2 matrices the amount increased for 7 days. The prolonged period of proliferation with TiO2 scaffolds resulted in a delay in alkaline phosphatase induction. However, osteocalcin incorporation into extracellular matrix by day 14 was greater with TiO2 scaffolds than with cpTi scaffolds. Calcium deposition was also greater with TiO2-coated substrates than with uncoated substrates. With the TiSi scaffolds osteocalcin production and mineralization were lower than with the cpTi scaffolds. The current study confirms our previous findings that titanium fiber mesh supports attachment, growth, and differentiation of rat bone marrow stromal cells. Furthermore, the osteogenic capacities of cell-scaffold constructs under cell culture conditions were increased with a sol-gel-derived titania coating, but not with a titania-silica coating.