Compaction enhances extracellular matrix content and mechanical properties of tissue-engineered cartilaginous constructs.
ABSTRACT: Many cell-based tissue-engineered cartilaginous constructs are mechanically softer than native tissue and have low content and abnormal proportions of extracellular matrix (ECM) constituents. We hypothesized that the load-bearing mechanical properties of cartilaginous constructs improve with the inclusion of collagen (COL) and proteoglycan (PG) during assembly. The objectives of this work were to determine (1) the effect of addition of PG, COL, or COL+PG on compressive properties of 2% agarose constructs and (2) the ability of mechanical compaction to concentrate matrix content and improve the compressive properties of such constructs. The inclusion of COL+PG improved the compressive properties of hydrogel constructs compared with PG or COL alone. Mechanical compaction increased the PG and COL concentrations in and compressive stiffness of the constructs. Chondrocytes included in the constructs maintained high viability after compaction. These results support the concepts that the assembly of cartilaginous constructs with COL+PG and application of mechanical compaction enhance the ECM content and compressive properties of engineered cartilaginous constructs.
Project description:Formation of tissue-engineered cartilage is greatly enhanced by mechanical stimulation. However, direct mechanical stimulation is not always a suitable method, and the utilization of mechanisms underlying mechanotransduction might allow for a highly effective and less aggressive alternate means of stimulation. In particular, the purinergic, adenosine 5'-triphosphate (ATP)-mediated signaling pathway is strongly implicated in mechanotransduction within the articular cartilage. We investigated the effects of transient and continuous exogenous ATP supplementation on mechanical properties of cartilaginous constructs engineered using bovine chondrocytes and human mesenchymal stem cells (hMSCs) encapsulated in an agarose hydrogel. For both cell types, we have observed significant increases in equilibrium and dynamic compressive moduli after transient ATP treatment applied in the fourth week of cultivation. Continuous ATP treatment over 4 weeks of culture only slightly improved the mechanical properties of the constructs, without major changes in the total glycosaminoglycan (GAG) and collagen content. Structure-function analyses showed that transiently ATP-treated constructs, and in particular those based on hMSCs, had the highest level of correlation between compositional and mechanical properties. Transiently treated groups showed intense staining of the territorial matrix for GAGs and collagen type II. These results indicate that transient ATP treatment can improve functional mechanical properties of cartilaginous constructs based on chondrogenic cells and agarose hydrogels, possibly by improving the structural organization of the bulk phase and territorial extracellular matrix (ECM), that is, by increasing correlation slopes between the content of the ECM components (GAG, collagen) and mechanical properties of the construct.
Project description:In vitro assembly of key functional extracellular matrix constituents for tissue-engineered constructs may provide a tool to modulate the retention of proteoglycan (PG) aggregates, which are crucial to compressive biomechanical properties of connective tissues. This study tested the hypotheses that (1) biomimetic molecular reassembly of PG aggregates (native aggrecan [AGC] with hyaluronan [HA] ± link protein [LP]) affects AGC retention kinetics in hydrogel constructs, (2) the compressive properties of such hydrogel constructs are related to the content of retained AGC, and (3) the reassembly method is compatible with chondrocytes. Addition of HA to AGC in hydrogel constructs increased AGC retention in a dose-dependent manner, and the addition of LP to AGC?+?HA further enhanced AGC retention. The level of AGC retention, in turn, was associated with increased equilibrium compressive stress of the constructs. Chondrocytes could be included in the process, and maintained expression of the chondrogenic phenotype, secreting type II collagen but little type I collagen. Thus, by altering the assembly of PG aggregates with HA ± LP, which affects AGC retention, it may be possible to achieve the targeted levels of PG components to modulate the mechanical properties of the engineered construct for cartilage as well as other tissues containing PG and PG aggregates.
Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling). Free-swelling MSC-seeded constructs were cultured for 21 days in chemically defined media. Chondrogenesis was induced with TGF-beta3. Undifferentiated controls were maintained in parallel. After 21 days of chondrogenic differentiation, a subset of constructs were subjected to 21 days of dynamic compressive loading. On days 21 and 42, construct mechanical properties and biochemical content were assessed. Microarray analysis was carried out on day 3, day 21 and day 42 constructs. 6 arrays.
Project description:Photopolymerizable and hydrolytically labile poly(ethylene glycol) (PEG) hydrogels formed from photo-clickable reactions were investigated as cell delivery platforms for cartilage tissue engineering (TE). PEG hydrogels were formed from thiol-norbornene PEG macromers whereby the crosslinks contained caprolactone segments with hydrolytically labile ester linkages. Juvenile bovine chondrocytes encapsulated in the hydrogels were cultured for up to four weeks and assessed biochemically and histologically, using standard destructive assays, and for mechanical and ultrasound properties, as nondestructive assays. Bulk degradation of acellular hydrogels was confirmed by a decrease in compressive modulus and an increase in mass swelling ratio over time. Chondrocytes deposited increasing amounts of sulfated glycosaminoglycans and collagens in the hydrogels with time. Spatially, collagen type II and aggrecan were present in the neotissue with formation of a territorial matrix beginning at day 21. Nondestructive measurements revealed an 8-fold increase in compressive modulus from days 7 to 28, which correlated with total collagen content. Ultrasound measurements revealed changes in the constructs over time, which differed from the mechanical properties, and appeared to correlate with ECM structure and organization shown by immunohistochemical analysis. Overall, non-destructive and destructive measurements show that this new hydrolytically degradable PEG hydrogel is promising for cartilage TE.Designing synthetic hydrogels whose degradation matches tissue growth is critical to maintaining mechanical integrity as the hydrogel degrades and new tissue forms, but is challenging due to the nature of the hydrogel crosslinks that inhibit diffusion of tissue matrix molecules. This study details a promising, new, photo-clickable and synthetic hydrogel whose degradation supports cartilaginous tissue matrix growth leading to the formation of a territorial matrix, concomitant with an increase in mechanical properties. Nondestructive assays based on mechanical and ultrasonic properties were also investigated using a novel instrument and found to correlate with matrix deposition and evolution. In sum, this study presents a new hydrogel platform combined with nondestructive assessments, which together have potential for in vitro cartilage tissue engineering.
Project description:Long-term dynamic compression enhanced the mechanical properties of MSC-seeded constructs only when loading was initiated after 21 days of chondrogenic differentiation. This study examined the molecular differences of chondrogenic MSCs compared to undifferentiated MSCs (TGF-beta vs no TGF-beta) and the effects of dynamic loading on MSC chondrogenesis (loading vs free-swelling). Overall design: Free-swelling MSC-seeded constructs were cultured for 21 days in chemically defined media. Chondrogenesis was induced with TGF-beta3. Undifferentiated controls were maintained in parallel. After 21 days of chondrogenic differentiation, a subset of constructs were subjected to 21 days of dynamic compressive loading. On days 21 and 42, construct mechanical properties and biochemical content were assessed. Microarray analysis was carried out on day 3, day 21 and day 42 constructs. 6 arrays.
Project description:Mesenchymal stem cells (MSCs) are an attractive cell source for cartilage tissue engineering and regenerative medicine. However, the use of these cells has been limited by their reduced ability to form functional tissue compared to chondrocytes when placed in three-dimensional culture systems. To optimize MSC functional chondrogenesis, we examined the effects of increasing seeding density and transient application of transforming growth factor beta 3 (TGF-beta3), two factors previously shown to improve growth of chondrocyte-based constructs. Chondrocytes seeded in agarose at 20 million cells/mL and MSCs seeded at 20 or 60 million cells/mL agarose were cultured for 7 weeks under continuous or transient application of TGF-beta3. In the transient group, cell-laden constructs were exposed to TGF-beta3 for the initial 3 weeks, followed by 4 weeks of culture in medium without TGF-beta3. Compressive properties, biochemical content, and gene expression were assessed at 3, 5, and 7 weeks. Matrix distribution and collagen type was determined using histology and immunohistochemistry, and chondrogenic and osteogenic markers were assessed using real-time polymerase chain reaction. When maintained continuously with TGF-beta3, chondrocyte-seeded constructs achieved a higher equilibrium compressive modulus than MSCs similarly maintained. Although properties of both groups increased with respect to starting values, there was no difference in bulk mechanical or biochemical properties with higher seeding density when MSCs were cultured with constant TGF-beta3. Findings also showed that while transient application of TGF-beta3 elicited robust growth from chondrocyte-laden gels, MSCs seeded at the same density failed to respond, although constructs maintained their previously accrued properties and continued to express cartilaginous genes after TGF-beta3 removal. Conversely, MSCs seeded at 60 million cells/mL exhibited a strong anabolic response with transient TGF-beta3 exposure, achieving an equilibrium modulus of approximately 200 kPa. Although this represents the highest modulus we have been able to achieve with MSC-seeded constructs using our culture system, further work remains to optimize MSC chondrogenesis for cartilage tissue engineering, particularly in terms of collagen content and dynamic mechanical properties.
Project description:The vertical vibration compaction method (VVCM), heavy compaction method and static pressure method were used to form phyllite specimens with different degrees of weathering. The influence of cement content, compactness, and compaction method on the mechanical properties of phyllite was studied. The mechanical properties of phyllite was evaluated in terms of unconfined compressive strength (Rc) and modulus of resilience (Ec). Further, test roads were paved along an expressway in China to demonstrate the feasibility of the highly weathered phyllite improvement technology. Results show that unweathered phyllite can be used as subgrade filler. In spite of increasing compactness, phyllite with a higher degree of weathering cannot meet the requirements for subgrade filler. With increasing cement content, Rc and Ec of the improved phyllite increases linearly. Rc and Ec increase by at least 15% and 17%, respectively, for every 1% increase in cement content and by at least 10% and 6%, respectively, for every 1% increase in compactness. The higher the degree of weathering of phyllite, the greater the degree of improvement of its mechanical properties.
Project description:Developing biomimetic cartilaginous tissues that support locomotion while maintaining chondrogenic behavior is a major challenge in the tissue engineering field. Specifically, while locomotive forces demand tissues with strong mechanical properties, chondrogenesis requires a soft microenvironment. To address this challenge, 3D cartilage-like tissue is bioprinted using two biomaterials with different mechanical properties: a hard biomaterial to reflect the macromechanical properties of native cartilage, and a soft biomaterial to create a chondrogenic microenvironment. To this end, a hard biomaterial (MPa order compressive modulus) composed of an interpenetrating polymer network (IPN) of polyethylene glycol (PEG) and alginate hydrogel is developed as an extracellular matrix (ECM) with self-healing properties, but low diffusive capacity. Within this bath supplemented with thrombin, fibrinogen containing human mesenchymal stem cell (hMSC) spheroids is bioprinted forming fibrin, as the soft biomaterial (kPa order compressive modulus) to simulate cartilage's pericellular matrix and allow a fast diffusion of nutrients. The bioprinted hMSC spheroids improve viability and chondrogenic-like behavior without adversely affecting the macromechanical properties of the tissue. Therefore, the ability to print locally soft and cell stimulating microenvironments inside of a mechanically robust hydrogel is demonstrated, thereby uncoupling the micro- and macromechanical properties of the 3D printed tissues such as cartilage.
Project description:Cartilage damage and/or aging effects can cause constant pain, which limits the patient's quality of life. Although different strategies have been proposed to enhance the limited regenerative capacity of cartilage tissue, the full production of native and functional cartilaginous extracellular matrix (ECM) has not yet been achieved. Poly(?-glutamic acid) (?-PGA), a naturally occurring polyamino acid, biodegradable into glutamate residues, has been explored for tissue regeneration. In this work, ?-PGA's ability to support the production of cartilaginous ECM by human bone marrow mesenchymal stem/stromal cells (MSCs) and nasal chondrocytes (NCs) was investigated. MSC and NC pellets were cultured in basal medium (BM), chondrogenic medium (CM), and CM-?-PGA-supplemented medium (CM+?-PGA) over a period of 21 days. Pellet size/shape was monitored with time. At 14 and 21 days of culture, the presence of sulfated glycosaminoglycans (sGAGs), type II collagen (Col II), Sox-9, aggrecan, type XI collagen (Col XI), type X collagen (Col X), calcium deposits, and type I collagen (Col I) was analyzed. After excluding ?-PGA's cytotoxicity, earlier cell condensation, higher sGAG content, Col II, Sox-9 (day 14), aggrecan, and Col X (day 14) production was observed in ?-PGA-supplemented MSC cultures, with no signs of mineralization or Col I. These effects were not evident with NCs. However, Sox-9 (at day 14) and Col X (at days 14 and 21) were increased, decreased, or absent, respectively. Overall, ?-PGA improved chondrogenic differentiation of MSCs, increasing ECM production earlier in culture. It is proposed that ?-PGA incorporation in novel biomaterials has a beneficial impact on future approaches for cartilage regeneration.
Project description:An abundant cell source is the cornerstone of most tissue engineering strategies, but extracting cells from the knee meniscus is hindered by its dense fibrocartilaginous matrix. Identifying a method to efficiently isolate meniscus cells is important, as it can reduce the cost and effort required to perform meniscus engineering research. In this study, six enzymatic digestion regimens used for cartilaginous cell isolation were used to isolate cells from the outer, middle, and inner regions of the bovine knee meniscus. Each regimen in each region was assessed in terms of cell yield, impact on cell phenotype, and cytotoxicity. All digestion regimens caused an overall upregulation of cartilage-specific genes Sox9, collagen type I (Col 1), collagen type II (Col 2), cartilage oligomeric matrix protein, and aggrecan (AGC) in cells from all meniscus regions, but was highest for cells isolated using 1075 U/mL of collagenase for 3 h (high collagenase). In response to isolation, outer meniscus cells showed highest upregulation of Sox9 and Col 1 genes, whereas greatest upregulation for middle meniscus cells was seen in Col 1 expression, and Col 2 expression for inner cells. Cell yield was highest in all regions when subjected to 45 min of 61 U/mL pronase followed by 3 h of 1075 U/mL collagenase (pronase/collagenase [P/C]) digestion regimen (outer: 6.57±0.37, middle: 12.77±1.41, inner: 22.17±1.47×10(6) cells/g tissue). The second highest cell yield was achieved using the low collagenase (LC) digestion regimen that applied 433 U/mL of collagenase for 18 h (outer: 1.95±0.54, middle: 3.3±4.4, inner: 6.06±2.44×10(6) cells/g tissue). Cytotoxicity analysis showed higher cell death in the LC group compared with the P/C group. Self-assembled constructs formed from LC-isolated cells were less dense than constructs formed from P/C-isolated cells, and P/C constructs showed higher glycosaminoglycan content and compressive moduli than LC constructs. All isolation methods tested resulted in similar phenotypic changes in meniscus cells from each region. These results indicate that, compared with other common isolation protocols, the P/C isolation method is able to more efficiently isolate meniscus cells from all regions that can produce tissue engineered constructs.