Amino Acid Uptake Limitations during Human Mesenchymal Stem Cell-Based Chondrogenesis.

A mino acids are the essential building blocks for collagen and proteoglycan, which are the main constituents for cartilage extracellular matrix (ECM). Synthesis of ECM proteins requires the uptake of various essential/nonessential amino acids. Analyzing amino acid metabolism during chondrogenesis can help to relate tissue quality to amino acid metabolism under different conditions. In our study, we studied amino acid uptake/secretion using human mesenchymal stem cell (hMSC)-based aggregate chondrogenesis in a serum-free induction medium with a defined chemical formulation. The initial glucose level and medium-change frequency were varied. Our results showed that essential amino acid uptake increased with time during hMSCs chondrogenesis for all initial glucose levels and medium-change frequencies. Essential amino acid uptake rates were initial glucose-level independent. The DNA-normalized glycosaminoglycans and hydroxyproline content of chondrogenic aggregates correlated with cumulative uptake of leucine, valine, and tryptophan regardless of initial glucose levels and medium-change frequencies. Collectively, our results show that amino acid uptake rates during in vitro chondrogenesis were insufficient to produce a tissue with an ECM content similar to that of human neonatal cartilage or adult cartilage. Furthermore, this deficiency was likely related to the downregulation of some key amino acid transporters in the cells. Such deficiency could be partially improved by increasing the amino acid availability in the chondrogenic medium by changing culture conditions.

Chondrogenesis of Mesenchymal Stem Cells through Local Release of TGF-ß3 from Heparinized Collagen Biofabric.

Osteoarthritic degeneration of cartilage is a major social health problem. Tissue engineering of cartilage using combinations of scaffold and mesenchymal stem cells (MSCs) is emerging as an alternative to existing treatment options such as microfracture, mosaicplasty, allograft, autologous chondrocyte implantation, or total joint replacement. Induction of chondrogenesis in high-density pellets of MSCs is generally attained by soluble exogenous TGF-ß3 in culture media, which requires lengthy in vitro culture period during which pellets gain mechanical robustness. On the other hand, a growth factor delivering and a mechanically robust scaffold material that can accommodate chondroid pellets would enable rapid deployment of pellets after seeding. Delivery of the growth factor from the scaffold locally would drive the induction of chondrogenic differentiation in the postimplantation period. Therefore, we sought to develop a biomaterial formulation that will induce chondrogenesis in situ, and compared its performance to soluble delivery in vitro. In this vein, a heparin-conjugated mechanically robust collagen fabric was developed for sustained delivery of TGF-ß3. The amount of conjugated heparin was varied to enhance the amount of TGF-ß3 uptake and release from the scaffold. The results showed that the scaffold delivered TGF-ß3 for up to 8 days of culture, which resulted in 15-fold increase in GAG production, and six-fold increase in collagen synthesis with respect to the No TGF-ß3 group. The resulting matrix was cartilage like, in that type II collagen and aggrecan were positive in the spheroids. Enhanced chondrogenesis under in situ TGF-ß3 administration resulted in a Young's modulus of ∼600 kPa. In most metrics, there were no significant differences between the soluble delivery group and in situ heparin-mediated delivery group. In conclusion, heparin-conjugated collagen scaffold developed in this study guides chondrogenic differentiation of hMSCs in a mechanically competent tissue construct, which showed potential to be used for cartilage tissue regeneration. Impact statement The most significant finding of this study was that sustained release of TGF-ß3 from heparinized collagen scaffold had chondroinductive effect on pelleted human mesenchymal stem cells (hMSCs). The effect was comparable to that observed in hMSC pellets that were cultured in chondrogenic media supplemented with TGF-ß3. The stiffness of scaffolds at the baseline was about 50% that of native cartilage and over 28 days the combined stiffness of pellet/scaffold complex converged to the stiffness of native cartilage. These data indicate that the scaffold system can generate a load-bearing cartilage-like tissue by using hMSCs pellets in a mechanically competent framework.

Glucose Availability Affects Extracellular Matrix Synthesis During Chondrogenesis In Vitro.

Understanding in vitro chondrogenesis of human mesenchymal stem cells (hMSCs) is important as it holds great promise for cartilage tissue engineering and other applications. The current technology produces the end tissue quality that is highly variable and dependent on culture conditions. We investigated the effect of nutrient availability on hMSC chondrogenesis in a static aggregate culture system by varying the medium-change frequency together with starting glucose levels. Glucose uptake and lactate secretion profiles were obtained to monitor the metabolism change during hMSC chondrogenesis with different culture conditions. Higher medium-change frequency led to increases in cumulative glucose uptake for all starting glucose levels. Furthermore, increase in glucose uptake by aggregates led to increased end tissue glycosaminoglycan (GAG) and hydroxyproline (HYP) content. The results suggest that increased glucose availability either through increased medium-change frequency or higher initial glucose levels lead to improved chondrogenesis. Also, cumulative glucose uptake and lactate secretion were found to correlate well with GAG and HYP content, indicating both molecules are promising biomarkers for noninvasive assessment of hMSC chondrogenesis. Collectively, our results can be used to design optimal culture conditions and develop dynamic assessment strategies for cartilage tissue engineering applications. Impact statement In this study, we investigated how culture conditions, medium-change frequency and glucose levels, affect chondrogenesis of human mesenchymal stem cells in an aggregate culture model. Doubling the medium-change frequency significantly increased the biochemical quality of the resultant tissue aggregates, as measured by their glycosaminoglycan and hydroxyproline content. We attribute this to increased glucose uptake through the glycolysis pathway, as secretion of lactate, a key endpoint product of the glycolysis pathway, increased concurrently. These findings can be used to design optimal culture conditions for tissue engineering and regenerative medicine applications.

Isolation of Chondrocytes from Human Cartilage and Cultures in Monolayer and 3D.

Chondrocytes are the only cell type in cartilage. The dense cartilage extracellular matrix surrounding the chondrocytes makes isolating these cells a complex and lengthy task that subjects the cells to harsh conditions. Protocols to isolate expand and maintain these cells have been improved over the years, providing ways to obtain viable cells for tissue engineering and clinical applications. Here we describe a method to obtain populations of chondrocytes that are able to expand and maintain a native-like phenotype.

The nonlinear relationship between speed of sound and compression in articular cartilage: Measurements and modeling.

We measured speed of sound in bovine articular cartilage as a function of compressive strain. Using techniques we developed, it was possible to apply strain starting from the unstrained, full height of a sample. Our measurements showed that speed of sound was not a monotonic function of strain as reported in earlier investigations. Speed increased with increasing strain over a range of lower strains. It reached a maximum, and then decreased as the strain increased further. These results were corroborated using a model of wave propagation in deformable porous materials. Using this model, we also established conditions under which a maximum in the speed would exist for samples in compression. Our measurements and analysis resolve the conflicting results reported in previous studies.

An Integrated Multi-Function Heterogeneous Biochemical Circuit for High-Resolution Electrochemistry-Based Genetic Analysis.

Modular construction of an autonomous and programmable multi-functional heterogeneous biochemical circuit that can identify, transform, translate, and amplify biological signals into physicochemical signals based on logic design principles can be a powerful means for the development of a variety of biotechnologies. To explore the conceptual validity, we design a CRISPR-array-mediated primer-exchange-reaction-based biochemical circuit cascade, which probes a specific biomolecular input, transform the input into a structurally accessible form for circuit wiring, translate the input information into an arbitrary sequence, and finally amplify the prescribed sequence through autonomous formation of a signaling concatemer. This upstream biochemical circuit is further wired with a downstream electrochemical interface, delivering an integrated bioanalytical platform. We program this platform to directly analyze the genome of SARS-CoV-2 in human cell lysate, demonstrating the capability and the utility of this unique integrated system.

Apparatus and Method for Rapid Detection of Acoustic Anisotropy in Cartilage.

PURPOSE: Articular cartilage is known to be mechanically anisotropic. In this paper, the acoustic anisotropy of bovine articular cartilage and the effects of freeze-thaw cycling on acoustic anisotropy were investigated. METHODS: We developed apparatus and methods that use a magnetic L-shaped sample holder, which allowed minimal handling of a tissue, reduced the number of measurements compared to previous studies, and produced highly reproducible results. RESULTS: SOS was greater in the direction perpendicular to the articular surface compared to the direction parallel to the articular surface (N=17, P = 0.00001). Average SOS was 1,758 ± 107 m/s perpendicular to the surface, and 1,617 ± 55 m/s parallel to it. The average percentage difference in SOS between the perpendicular and parallel directions was 8.2% (95% CI: 5.4% to 11%). Freeze-thaw cycling did not have a significant effect on SOS (P>0.4). CONCLUSION: Acoustic measurement of tissue properties is particularly attractive for work in our laboratory since it has the potential for nondestructive characterization of the properties of developing engineered cartilage. Our approach allowed us to observe acoustic anisotropy of articular cartilage rapidly and reproducibly. This property was not significantly affected by freeze-thawing of the tissue samples, making cryopreservation practical for these assays.

Exploring the Trans-Cleavage Activity of CRISPR-Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor.

An accurate, rapid, and cost-effective biosensor for the quantification of disease biomarkers is vital for the development of early-diagnostic point-of-care systems. The recent discovery of the trans-cleavage property of CRISPR type V effectors makes CRISPR a potential high-accuracy bio-recognition tool. Herein, a CRISPR-Cas12a (cpf1) based electrochemical biosensor (E-CRISPR) is reported, which is more cost-effective and portable than optical-transduction-based biosensors. Through optimizing the in vitro trans-cleavage activity of Cas12a, E-CRIPSR was used to detect viral nucleic acids, including human papillomavirus 16 (HPV-16) and parvovirus B19 (PB-19), with a picomolar sensitivity. An aptamer-based E-CRISPR cascade was further designed for the detection of transforming growth factor ß1 (TGF-ß1) protein in clinical samples. As demonstrated, E-CRISPR could enable the development of portable, accurate, and cost-effective point-of-care diagnostic systems.

Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three-dimensionally woven scaffolds.

The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three-dimensionally woven poly(ε-caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations-PCL alone, PCL-bone, "point-of-care" seeded MSC-PCL-bone, and chondrogenically precultured Ch-MSC-PCL-bone constructs-were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch-MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.

Rapid Detection of Shear-Induced Damage in Tissue-Engineered Cartilage Using Ultrasound.

Previous investigations have shown that tissue-engineered articular cartilage can be damaged under a combination of compression and sliding shear. In these cases, damage was identified in histological sections after a test was completed. This approach is limited, in that it does not identify when damage occurred. This especially limits the utility of an assay for evaluating damage when comparing modifications to a tissue-engineering protocol. In this investigation, the feasibility of using ultrasound (US) to detect damage as it occurs was investigated. US signals were acquired before, during, and after sliding shear, as were stereomicroscope images of the cartilage surface. Histology was used as the standard for showing if a sample was damaged. We showed that US reflections from the surface of the cartilage were attenuated due to roughening following sliding shear. Furthermore, it was shown that by scanning the transducer across a sample, surface roughness and erosion following sliding shear could be identified. Internal delamination could be identified by the appearance of new echoes between those from the front and back of the sample. Thus, it is feasible to detect damage in engineered cartilage using US.

Dynamics of Intrinsic Glucose Uptake Kinetics in Human Mesenchymal Stem Cells During Chondrogenesis.

Chondrogenesis of human mesenchymal stem cells (hMSCs) is an important biological process in many applications including cartilage tissue engineering. We investigated the glucose uptake characteristics of aggregates of hMSCs undergoing chondrogenesis over a 3-week period both experimentally and by using a mathematical model. Initial concentrations of glucose in the medium were varied from 1 to 4.5 g/L to mimic limiting conditions and glucose uptake profiles were obtained. A reaction-diffusion mathematical model was implemented and solved to estimate kinetic parameters. Experimental glucose uptake rates increased with culture time for aggregates treated with higher initial glucose concentrations (3 and 4.5 g/L), whereas they decreased or remained constant for those treated with lower initial glucose concentrations (1 and 2 g/L). Lactate production rate increased by as much as 40% for aggregates treated with higher initial glucose concentrations (2, 3 and 4.5 g/L), whereas it remained constant for those treated with 1 g/L initial glucose concentration. The estimated DNA-normalized maximum glucose uptake rate decreased by a factor of 9 from day 0-2 (12.5 mmol/s/g DNA) to day 6-8 (1.5 mmol/s/g DNA), after which it started to increase. On day 18-20, its value (17.5 mmol/s/g DNA) was about 11 times greater than its lowest value. Further, the extracellular matrix levels of aggregates at day 14 and day 21 correlated with their overall glucose uptake and lactate production. The results suggest that during chondrogenesis, for optimal results, cells require increasing amounts of glucose. Our results also suggest that diffusion limitations play an important role in glucose uptake even in the smaller size aggregate model of chondrogenesis. Further, the results indicate that glucose uptake or lactate production can be a tool for predicting the end quality of tissue during the process of chondrogenesis. The estimated kinetic parameters can be used to model glucose requirements in cartilage tissue engineering applications.

ROCK Inhibition Promotes the Development of Chondrogenic Tissue by Improved Mass Transport.

Human mesenchymal stem cell (hMSC)-based chondrogenesis is a key process used to develop tissue engineered cartilage constructs from stem cells, but the resulting constructs have inferior biochemical and biomechanical properties compared to native articular cartilage. Transforming growth factor ß containing medium is commonly applied to cell layers of hMSCs, which aggregate upon centrifugation to form 3-D constructs. The aggregation process leads to a high cell density condition, which can cause nutrient limitations during long-term culture and, subsequently, inferior quality of tissue engineered constructs. Our objective is to modulate the aggregation process by targeting RhoA/ROCK signaling pathway, the chief modulator of actomyosin contractility, to enhance the end quality of the engineered constructs. Through ROCK inhibition, repression of cytoskeletal tension in chondrogenic hMSCs was achieved along with less dense aggregates with enhanced transport properties. ROCK inhibition also led to significantly increased cartilaginous extracellular matrix accumulation. These findings can be used to create an improved microenvironment for hMSC-derived tissue engineered cartilage culture. We expect that these findings will ultimately lead to improved cartilaginous tissue development from hMSCs.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function.

Expression of miR-145-5p During Chondrogenesis of Mesenchymal Stem Cells.

Assessing the quality of tissue engineered (TE) cartilage has historically been performed by endpoint measurements including marker gene expression. Until the adoption of promoter-driven reporter constructs capable of quantitative and real time non-destructive expression analysis, temporal gene expression assessments along a timeline could not be performed on TE constructs. We further exploit this technique to utilize microRNA (miRNA or miR) through the use of firefly luciferase reporter (Luc) containing a 3' UTR perfect complementary target sequence to the mature miR-145-5p. We report the development and testing of a firefly luciferase (Luc) reporter responsive to miR-145-5p for longitudinal tracking of miR-145-5p expression throughout MSC chondrogenic differentiation. Plasmid reporter vectors containing a miR-145-5p responsive reporter (Luc reporter with a perfect complementary target sequence to the mature miR-145-5p sequence in the 3'UTR), a Luc reporter driven by a truncated Sox9 (one of the targets of miR-145-5p) promoter, or the Luc backbone (control) vector without a specific miRNA target were transfected into MSCs by electroporation. Transfected MSCs were mixed with untransfected MSC to generate chondrogenic pellets. Pellets were imaged by bioluminescent imaging (BLI) and harvested along a preset time line. The imaging signals from miR-145-5p responsive reporter and Sox9 promoter-driven reporter showed correlated time-courses (measured by BLI and normalized to Luc-control reporter; Spearman r=0.93, p=0.0002) during MSC chondrogenic differentiation. Expression analysis by qRT-PCR suggests an inverse relationship between miR-145-5p and Sox9 gene expression during MSC chondrogenic differentiation. Non-destructive cell-pellet imaging is capable of supplementing histological analyses to characterize TE cartilage. The miR-145-5p responsive reporter is relatively simple to construct and generates a consistent imaging signal responsive to miR-145-5p during MSC chondrogenesis in parallel to certain molecular and cellular events.

Growth Factor Dose Tuning for Bone Progenitor Cell Proliferation and Differentiation on Resorbable Poly(propylene fumarate) Scaffolds.

One approach to the development of an artificial graft material could rely on uniform coverage of a resorbable biomaterial with bone extracellular matrix (ECM). To achieve this on the surface of poly(propylene fumarate) (PPF) scaffolds, we selected a growth factor regime of basic fibroblast growth factor (FGF-2) (5 ng/mL), platelet-derived growth factor (PDGF-BB) (40 ng/mL), and epidermal growth factor (EGF) (20 ng/mL) to stimulate proliferation of bone marrow-derived human mesenchymal stem cells (BM-hMSCs). Bone morphogenetic protein (BMP) 4 (50 ng/mL), 6 (50 ng/mL), and 7 (27 ng/mL) in the presence of the following osteogenic substances: dexamethasone (10(-7) M), ß-glycerophosphate (10 mM), and ascorbic acid (50 µg/mL) were chosen to induce differentiation of BM-hMSCs into ECM-secreting osteoblasts. These growth factors were also studied at 10× concentration to determine dose effect. Proliferation was analyzed by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, scanning electron microscopy (SEM), and toluidine blue staining, whereas differentiation was analyzed through alizarin red S staining and assay, alkaline phosphatase (ALP) staining and assay, and SEM. The proliferation study suggests that a combination of EGF, PDGF-BB, and FGF-2 growth factors at optimal concentration over a period of 1 week exhibits significantly (p = 0.001) higher number of cells (116,024 ± 5165) than these cytokines without EGF (91,706 ± 11,965). Increasing the dosage does not show any significant effect. The BM-hMSC differentiation study results show that ALP enzyme production and mineral deposition increase from day 14 to day 21 in all groups containing BMPs and osteogenic medium. However, mineralization is significantly higher in the BMP-7 group. Furthermore, the feasibility of translating the results from two dimensional thin films to three dimensional-printed PPF scaffolds was determined through uniform initial seeding and spreading of BM-hMSCs. Therefore, we have determined the optimum dose of growth factors for proliferation and differentiation of BM-hMSCs on the surface of PPF scaffolds, which can be used to produce ECM-coated implants for the treatment of bone defects.

Nondestructive Techniques to Evaluate the Characteristics and Development of Engineered Cartilage.

In this review, methods for evaluating the properties of tissue engineered (TE) cartilage are described. Many of these have been developed for evaluating properties of native and osteoarthritic articular cartilage. However, with the increasing interest in engineering cartilage, specialized methods are needed for nondestructive evaluation of tissue while it is developing and after it is implanted. Such methods are needed, in part, due to the large inter- and intra-donor variability in the performance of the cellular component of the tissue, which remains a barrier to delivering reliable TE cartilage for implantation. Using conventional destructive tests, such variability makes it near-impossible to predict the timing and outcome of the tissue engineering process at the level of a specific piece of engineered tissue and also makes it difficult to assess the impact of changing tissue engineering regimens. While it is clear that the true test of engineered cartilage is its performance after it is implanted, correlation of pre and post implantation properties determined non-destructively in vitro and/or in vivo with performance should lead to predictive methods to improve quality-control and to minimize the chances of implanting inferior tissue.

Ultrasound Elastography for Estimation of Regional Strain of Multilayered Hydrogels and Tissue-Engineered Cartilage.

Tissue-engineered (TE) cartilage constructs tend to develop inhomogeneously, thus, to predict the mechanical performance of the tissue, conventional biomechanical testing, which yields average material properties, is of limited value. Rather, techniques for evaluating regional and depth-dependent properties of TE cartilage, preferably non-destructively, are required. The purpose of this study was to build upon our previous results and to investigate the feasibility of using ultrasound elastography to non-destructively assess the depth-dependent biomechanical characteristics of TE cartilage while in a sterile bioreactor. As a proof-of-concept, and to standardize an assessment protocol, a well-characterized three-layered hydrogel construct was used as a surrogate for TE cartilage, and was studied under controlled incremental compressions. The strain field of the construct predicted by elastography was then validated by comparison with a poroelastic finite-element analysis (FEA). On average, the differences between the strains predicted by elastography and the FEA were within 10%. Subsequently engineered cartilage tissue was evaluated in the same test fixture. Results from these examinations showed internal regions where the local strain was 1-2 orders of magnitude greater than that near the surface. These studies document the feasibility of using ultrasound to evaluate the mechanical behaviors of maturing TE constructs in a sterile environment.

Towards the feasibility of using ultrasound to determine mechanical properties of tissues in a bioreactor.

Our ultimate goal is to non-destructively evaluate mechanical properties of tissue-engineered (TE) cartilage using ultrasound (US). We used agarose gels as surrogates for TE cartilage. Previously, we showed that mechanical properties measured using conventional methods were related to those measured using US, which suggested a way to non-destructively predict mechanical properties of samples with known volume fractions. In this study, we sought to determine whether the mechanical properties of samples, with unknown volume fractions could be predicted by US. Aggregate moduli were calculated for hydrogels as a function of SOS, based on concentration and density using a poroelastic model. The data were used to train a statistical model, which we then used to predict volume fractions and mechanical properties of unknown samples. Young's and storage moduli were measured mechanically. The statistical model generally predicted the Young's moduli in compression to within <10% of their mechanically measured value. We defined positive linear correlations between the aggregate modulus predicted from US and both the storage and Young's moduli determined from mechanical tests. Mechanical properties of hydrogels with unknown volume fractions can be predicted successfully from US measurements. This method has the potential to predict mechanical properties of TE cartilage non-destructively in a bioreactor.

Chondrogenic differentiation of mesenchymal stem cells: challenges and unfulfilled expectations.

Articular cartilage repair and regeneration provides a substantial challenge in Regenerative Medicine because of the high degree of morphological and mechanical complexity intrinsic to hyaline cartilage due, in part, to its extracellular matrix. Cartilage remains one of the most difficult tissues to heal; even state-of-the-art regenerative medicine technology cannot yet provide authentic cartilage resurfacing. Mesenchymal stem cells (MSCs) were once believed to be the panacea for cartilage repair and regeneration, but despite years of research, they have not fulfilled these expectations. It has been observed that MSCs have an intrinsic differentiation program reminiscent of endochondral bone formation, which they follow after exposure to specific reagents as a part of current differentiation protocols. Efforts have been made to avoid the resulting hypertrophic fate of MSCs; however, so far, none of these has recreated a fully functional articular hyaline cartilage without chondrocytes exhibiting a hypertrophic phenotype. We reviewed the current literature in an attempt to understand why MSCs have failed to regenerate articular cartilage. The challenges that must be overcome before MSC-based tissue engineering can become a front-line technology for successful articular cartilage regeneration are highlighted.

Validating continuous digital light processing (cDLP) additive manufacturing accuracy and tissue engineering utility of a dye-initiator package.

This study tested the accuracy of tissue engineering scaffold rendering via the continuous digital light processing (cDLP) light-based additive manufacturing technology. High accuracy (i.e., <50 µm) allows the designed performance of features relevant to three scale spaces: cell-scaffold, scaffold-tissue, and tissue-organ interactions. The biodegradable polymer poly (propylene fumarate) was used to render highly accurate scaffolds through the use of a dye-initiator package, TiO2 and bis (2,4,6-trimethylbenzoyl)phenylphosphine oxide. This dye-initiator package facilitates high accuracy in the Z dimension. Linear, round, and right-angle features were measured to gauge accuracy. Most features showed accuracies between 5.4-15% of the design. However, one feature, an 800 µm diameter circular pore, exhibited a 35.7% average reduction of patency. Light scattered in the x, y directions by the dye may have reduced this feature's accuracy. Our new fine-grained understanding of accuracy could be used to make further improvements by including corrections in the scaffold design software. Successful cell attachment occurred with both canine and human mesenchymal stem cells (MSCs). Highly accurate cDLP scaffold rendering is critical to the design of scaffolds that both guide bone regeneration and that fully resorb. Scaffold resorption must occur for regenerated bone to be remodeled and, thereby, achieve optimal strength.