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.

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.

Multimodal evaluation of tissue-engineered cartilage.

Tissue engineering (TE) has promise as a biological solution and a disease modifying treatment for arthritis. Although cartilage can be generated by TE, substantial inter- and intra-donor variability makes it impossible to guarantee optimal, reproducible results. TE cartilage must be able to perform the functions of native tissue, thus mechanical and biological properties approaching those of native cartilage are likely a pre-requisite for successful implantation. A quality-control assessment of these properties should be part of the implantation release criteria for TE cartilage. Release criteria should certify that selected tissue properties have reached certain target ranges, and should be predictive of the likelihood of success of an implant in vivo. Unfortunately, it is not currently known which properties are needed to establish release criteria, nor how close one has to be to the properties of native cartilage to achieve success. Achieving properties approaching those of native cartilage requires a clear understanding of the target properties and reproducible assessment methodology. Here, we review several main aspects of quality control as it applies to TE cartilage. This includes a look at known mechanical and biological properties of native cartilage, which should be the target in engineered tissues. We also present an overview of the state of the art of tissue assessment, focusing on native articular and TE cartilage. Finally, we review the arguments for developing and validating non-destructive testing methods for assessing TE products.

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.

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.

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.

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.

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.

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.

Simplification of aggregate culture of human mesenchymal stem cells as a chondrogenic screening assay.

Aggregate culture provides a three-dimensional (3-D) environment for differentiating or differentiated cells; it is particularly useful to study in vitro chondrogenesis and cartilage biology. We have recently ported this method from a conical tube-based format to a 96-well plate format for the study of mesenchymal stem cell (MSC) chondrogenesis. The microplate format has greatly reduced the workload and materials cost, while maintaining reproducible chondrogenic differentiation. A long-term goal is to fully automate aggregate culture--this requires critically identifying all the indispensable steps of the protocol. Robotic laboratory equipment for manipulating microplate assays are commercially available; however centrifugation steps are difficult to implement automatically. We, therefore, tested whether the centrifugation step can be eliminated, thus significantly streamlining the assay workflow. By comparing aggregates prepared from human bone marrow-derived MSCs (hMSCs) that were formed either through centrifugation or through free sedimentation, we found that both methods produce aggregates with similar formation kinetics, and that there was no perceptible difference in the timing of the appearance of markers of chondrogenesis. Thus, it appears safe to eliminate the centrifugation step from the aggregate culture protocol. This results in significant time and effort savings and paves the way for future full automation of the aggregate assay.

Cartilage tissue engineering for laryngotracheal reconstruction: comparison of chondrocytes from three anatomic locations in the rabbit.

Tissue engineering may provide a technique to generate cartilage grafts for laryngotracheal reconstruction in children. The present study used a rabbit model to characterize cartilage generated by a candidate tissue engineering approach to determine, under baseline conditions, which chondrocytes in the rabbit produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. We characterized tissue-engineered cartilage generated in perfused bioreactor chambers from three sources of rabbit chondrocytes: articular, auricular, and nasal cartilage. Biomechanical testing and histological, immunohistochemical, and biochemical assays were performed to determine equilibrium unconfined compression (Young's) modulus, and biochemical composition and structure. We found that cartilage samples generated from articular or nasal chondrocytes lacked the mechanical integrity and stiffness necessary for completion of the biomechanical testing, but five of six auricular samples completed the biomechanical testing (moduli of 210 +/- 93 kPa in two samples at 3 weeks and 100 +/- 65 kPa in three samples at 6 weeks). Auricular samples showed more consistent staining for proteoglycans and collagen II and had significantly higher glycosaminoglycan (GAG) content and concentration and higher collagen content than articular or nasal samples. In addition, the delayed gadolinium enhanced MRI of cartilage (dGEMRIC) method revealed variations in GAG spatial distribution in auricular samples that were not present in articular or nasal samples. The results indicate that, for the candidate tissue engineering approach under baseline conditions, only rabbit auricular chondrocytes produce tissue-engineered cartilage suitable for in vivo testing in laryngotracheal reconstruction. The results also suggest that this and similar tissue engineering approaches must be optimized for each potential source of chondrocytes.

Evaluation of machining methods for trabecular metal implants in a rabbit intramedullary osseointegration model.

Implant success is dependent in part on the interaction of the implant with the surrounding tissues. Porous tantalum implants (Trabecular Metal, TM) have been shown to have excellent osseointegration. Machining this material to complex shapes with close tolerances is difficult because of its open structure and the ductile nature of metallic tantalum. Conventional machining results in occlusion of most of the surface porosity by the smearing of soft metal. This study compared TM samples finished by three processing techniques: conventional machining, electrical discharge machining, and nonmachined, "as-prepared." The TM samples were studied in a rabbit distal femoral intramedullary osseointegration model and in cell culture. We assessed the effects of these machining methods at 4, 8, and 12 weeks after implant placement. The finishing technique had a profound effect on the physical presentation of the implant interface: conventional machining reduced surface porosity to 30% compared to bulk porosities in the 70% range. Bone ongrowth was similar in all groups, while bone ingrowth was significantly greater in the nonmachined samples. The resulting mechanical properties of the bone implant-interface were similar in all three groups, with only interface stiffness and interface shear modulus being significantly higher in the machined samples.

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.

Hydrostatic pressure increases apoptosis in cartilage-constructs produced from human osteoarthritic chondrocytes.

Tissue-engineering is considered a promising avenue for developing human articular cartilage implants that can be employed for resurfacing damaged cartilage in the early stages of osteoarthritis. In the present study, human cartilage-constructs were produced from human osteoarthritic chondrocytes maintained on a scaffold of HYAFFR-11 in perfusion mini-bioreactors or after implantation and recovery from nude or SCID mice after 3 weeks. The human cartilage-construct extracellular matrix reacted positively with anti-Type II collagen monoclonal antibody, but not with anti-Type I or anti-Type X collagen monoclonal antibodies. A significant portion of the cartilage-construct extracellular matrix stained metachromatic with Toluidine blue-O indicative of sulfated-proteoglycan deposition. Cyclic hydrostatic pressure applied for 4 hrs at 5 MPa using a 1 Hertz sinusoidal frequency significantly increased (p < 0.02) the proportion of apoptotic cells in the cartilage-constructs (41% +/- 4.2%; mean +/- SD) compared to control cartilage-constructs (28.5 +/- 8.4%).

A rapid seeding technique for the assembly of large cell/scaffold composite constructs.

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.