Hyaluronic acid facilitates chondrogenesis and matrix deposition of human adipose derived mesenchymal stem cells and human chondrocytes co-cultures.

Clinical success on cartilage regeneration could be achieved by using available biomaterials and cell-based approaches. In this study, we have developed a composite gel based on collagen/hyaluronic acid (Coll-HA) as ideal, physiologically representative 3D support for in vitro chondrogenesis of human adipose-derived mesenchymal stem cells (hAMSCs) co-cultured with human articular chondrocytes (hAC). The incorporation of hyaluronic acid (HA) attempted to provide an additional stimulus to the hAMSCs for chondrogenesis and extracellular matrix deposition. Coll-HA gels were fabricated by directly mixing different amounts of HA (0-5%) into collagen solution before gelation. hACs and hAMSCs were co-cultured at different ratios from 100% to 0% in steps of 25%. Thus, five different co-culture groups were tested in the various Coll-HA 3D matrices. HA greatly impacted the cell viability and proliferation as well as the mechanical properties of the Coll-HA gel. The effective Young's modulus changed from 5.8 to 9.0kPa with increasing concentrations of HA in the gel. In addition, significantly higher amounts of glycosaminoglycan (GAG) were detected that seemed to be dependent on HA content. The highest HA concentration used (5%) resulted in the lowest Collagen type X (Col10) expression for most of the cell culture groups. Unexpectedly, culturing in these gels was also associated with decreased SOX9 and Collagen type II (Col2) expression, while Collagen type III (Col3) and metalloproteinase 13 notably increased. By using 1% HA, a positive effect on SOX9 expression was observed in the co-culture groups. In addition, a significant increase in GAGs production was also detected. Regarding co-culturing, the group with 25% hAMSCs+75% hACs was the most chondrogenic one considering SOX9 and Col2 expression as well as GAGs production. This group showed negligible Col10 expression after 35days of culture independently of the gel used. It also featured the highest effective Young's modulus (9.9kPa) when cultivated in the 1% HA matrix. Concerning the level of dissolved oxygen in situ, the groups with a higher amount of hAMSCs showed lower oxygen levels (40-58% O2) compared to hACs (63-74% O2). This might be attributed to the higher cellular metabolism and proliferation rate of the hAMSCs. Interestingly, lower oxygen was detected in the HA-containing gels when compared to plain collagen. This may contribute to the better chondrogenesis observed in these groups. Altogether, our results indicated that HA may favor chondrogenesis, but its effect highly depends on the concentration used. Additionally, co-culture of hACs with hAMSCs also favors chondrogenesis and especially increases extracellular matrix production and decreases hypertrophy. STATEMENT OF SIGNIFICANCE: In the clinical situation, large cartilage defects can be treated with MACT. However, this is a two-stage procedure, which increases the risk for the patient. Moreover, culturing chondrocytes leads to dedifferentiation. The matrix used for MACT is a collagen-based scaffold. In this study, it was demonstrated that hyaluronic acid, a natural component of the extracellular matrix, supplementation to a collagen hydrogel stimulates chondrogenic differentiation in a dose dependent manner. 1% HA showed the best overall results. Furthermore, exchanging 25% of human articular chondrocytes with adipose-derived mesenchymal stem cells didn't change the chondrogenic potential, but reduced going in unwanted pathways and improved biomechanical properties. This could translate to a one-step procedure and shows the potential of inducing differentiation by natural biomaterials.

Mimicking the cardiac cycle in intact cardiomyocytes using diastolic and systolic force clamps; measuring power output.

AIMS: A single isolated cardiomyocyte is the smallest functional unit of the heart. Yet, all single isolated cardiomyocyte experiments have been limited by the lack of proper methods that could reproduce a physiological cardiac cycle. We aimed to investigate the contractile properties of a single cardiomyocyte that correctly mimic the cardiac cycle. METHODS AND RESULTS: By adjusting the parameters of the feedback loop, using a suitably engineered feedback system and recording the developed force and the length of a single rat cardiomyocyte during contraction and relaxation, we were able to construct force-length (FL) relations analogous to the pressure-volume (PV) relations at the whole heart level. From the cardiac loop graphs, we obtained, for the first time, the power generated by one single cardiomyocyte. CONCLUSION: Here, we introduce a new approach that by combining mechanics, electronics, and a new type optical force transducer can measure the FL relationship of a single isolated cardiomyocyte undergoing a mechanical loop that mimics the PV cycle of a beating heart.

Platelet Control of Fibrin Distribution and Microelasticity in Thrombus Formation Under Flow.

OBJECTIVE: Platelet- and fibrin-dependent thrombus formation is regulated by blood flow and exposure of collagen and tissue factor. However, interactions between these blood-borne and vascular components are not well understood. APPROACH AND RESULTS: Here, we developed a method to assess whole-blood thrombus formation on microspots with defined amounts of collagen and tissue factor, allowing determination of the mechanical properties and intrathrombus composition. Confining the collagen content resulted in diminished platelet deposition and fibrin formation at high shear flow conditions, but this effect was compensated by a larger thrombus size and increased accumulation of fibrin in the luminal regions of the thrombi at the expense of the base regions. These thrombi were more dependent on tissue factor-triggered thrombin generation. Microforce nanoindentation analysis revealed a significantly increased microelasticity of thrombi with luminal-oriented fibrin. At a low shear rate, fibrin fibers tended to luminally cover the thrombi, again resulting in a higher microelasticity. Studies with blood from patients with distinct hemostatic insufficiencies indicated an impairment in the formation of a platelet-fibrin thrombus in the cases of dilutional coagulopathy, thrombocytopenia, Scott syndrome, and hemophilia B. CONCLUSIONS: Taken together, our data indicate that (1) thrombin increases the platelet thrombus volume; (2) tissue factor drives the formation of fibrin outside of the platelet thrombus; (3) limitation of platelet adhesion redirects fibrin from bottom to top of the thrombus; (4) a lower shear rate promotes thrombus coverage with fibrin; (5) the fibrin distribution pattern determines thrombus microelasticity; and (6) the thrombus-forming process is reduced in patients with diverse hemostatic defects.