An official website of the United States government
Articular cartilage is comprised of two main components, the extracellular matrix (ECM) and the pericellular matrix (PCM). The PCM helps to protect chondrocytes in the cartilage from mechanical loads, but in patients with osteoarthritis, the PCM is weakened, resulting in increased chondrocyte stress. As chondrocytes are responsible for matrix synthesis and maintenance, it is important to understand how mechanical loads affect the cellular responses of chondrocytes. Many studies have examined chondrocyte responses to in vitro mechanical loading by embedding chondrocytes in 3-D hydrogels. However, these experiments are mostly performed in the absence of PCM, which may obscure important responses to mechanotransduction. Here, drop-based microfluidics is used to culture single chondrocytes in alginate microgels for cell-directed PCM synthesis that closely mimics the in vivo microenvironment. Chondrocytes formed PCM over 10 days in these single-cell 3-D microenvironments. Mechanotransduction studies were performed, in which single-cell microgels mimicking the cartilage PCM were embedded in high-stiffness agarose. After physiological dynamic compression in a custom-built bioreactor, microgels exhibited distinct metabolomic profiles from both uncompressed and monolayer controls. These results demonstrate the potential of single cell encapsulation in alginate microgels to advance cartilage tissue engineering and basic chondrocyte mechanobiology.
more »
« less
Characterization of Composite Agarose–Collagen Hydrogels for Chondrocyte Culture
Abstract To elucidate the mechanisms of cellular mechanotransduction, it is necessary to employ biomaterials that effectively merge biofunctionality with appropriate mechanical characteristics. Agarose and collagen separately are common biopolymers used in cartilage mechanobiology and mechanotransduction studies but lack features that make them ideal for functional engineered cartilage. In this study, agarose is blended with collagen type I to create hydrogels with final concentrations of 4% w/v or 2% w/v agarose with 2 mg/mL collagen. We hypothesized that the addition of collagen into a high-concentration agarose hydrogel does not diminish mechanical properties. Acellular and cell-laden studies were completed to assess rheologic and compressive properties, contraction, and structural homogeneity in addition to cell proliferation and sulfated glycosaminoglycan production. Over 21 days in culture, cellular 4% agarose–2 mg/mL collagen I hydrogels seeded with primary murine chondrocytes displayed structural and bulk mechanical behaviors that did not significantly alter from 4% agarose-only hydrogels, cell proliferation, and continual glycosaminoglycan production, indicating promise toward the development of an effective hydrogel for chondrocyte mechanotransduction and mechanobiology studies.
more »
« less
- Award ID(s):
- 2149946
- PAR ID:
- 10541814
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Annals of Biomedical Engineering
- Volume:
- 53
- Issue:
- 1
- ISSN:
- 0090-6964
- Format(s):
- Medium: X Size: p. 120-132
- Size(s):
- p. 120-132
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Bulk measurements of ECM stiffness are commonly used in mechanobiology. However, peri-cellular stiffness can be quite heterogenous and divergent from the bulk properties. Here, we use optical tweezers active microrheology (AMR) to quantify how two different cell lines embedded in 1.0 and 1.5 mg/ml type 1 collagen (T1C) establish dissimilar patterns of peri-cellular stiffness. We found that dermal fibroblasts (DFs) increase local stiffness of 1.0 mg/ml T1C hydrogels, but surprisingly do not alter stiffness of 1.5 mg/ml T1C hydrogels. In contrast, MDA-MB-231 cells (MDAs) predominantly do not stiffen T1C hydrogels, as compared to cell-free controls. Results suggest that MDAs adapt to the bulk ECM stiffness, while DFs regulate local stiffness to levels they intrinsically “prefer”. Further, cells were subjected to treatments, that were previously shown to alter migration, proliferation and contractility of DFs and MDAs. Following treatment, both cell lines established different levels of stiffness magnitude and anisotropy, which were dependent on the cell line, T1C concentration and treatment. In summary, our findings demonstrate that AMR reveals otherwise masked mechanical properties such as spatial gradients and anisotropy, which are known to affect cell behavior at the macro-scale.more » « less
-
Bulk ECM stiffness measurements are often used in research on cell mechanobiology. However, past studies by our group have shown that peri-cellular stiffness can span few orders of magnitude and diverges from the bulk properties. Us- ing optical tweezers active microrheology (AMR) we can de- scribe stiffness landscape around individual cells. In this study, we show how different cell lines cultured in 1.0 and 1.5 mg/ml type 1 collagen (T1C) create disparate patterns of peri-cellular stiffness. Dermal fibroblasts (DFs) increase peri-cellular stiffness, when embedded in 1.0 mg/ml T1C hy- drogels, but do not alter stiffness in 1.5 mg/ml T1C hydro- gels. In contrast, invasive human breast cancer MDA-MB- 231 cells (MDAs) do not significantly change the stiffness of T1C hydrogels, as compared to cell-free controls. Results indicate that while MDAs adapt to the bulk ECM stiffness, DFs regulate local stiffness to levels they intrinsically “fa- vor”. Further, cells were also subjected to treatments that were previously shown to regulate their migration, prolifera- tion and contractility. Following each treatment, cells estab- lished dissimilar stiffness patterns. Stiffness magnitude and extent of anisotropy varied with the cell line, T1C concen- tration and treatment. In summary, we demonstrate that AMR can reveal otherwise masked mechanical properties of the local ECM, which are known to affect cell behavior.more » « less
-
Abstract A majority of breast cancer deaths occur due to metastasis of cancer cells to distant organs. In particular, brain metastasis is very aggressive with an extremely low survival rate. Breast cancer cells that metastasize to the brain can enter a state of dormancy, which allows them to evade death. The brain microenvironment provides biophysical, biochemical, and cellular cues, and plays an important role in determining the fate of dormant cancer cells. However, how these cues influence dormancy remains poorly understood. Herein, we employed hyaluronic acid (HA) hydrogels with a stiffness of ~0.4 kPa as an in vitro biomimetic platform to investigate the impact of biochemical cues, specifically alterations in RGD concentration, on dormancy versus proliferation in MDA‐MB‐231Br brain metastatic breast cancer cells. We applied varying concentrations of RGD peptide (0, 1, 2, or 4 mg/mL) to HA hydrogel surfaces and confirmed varying degrees of surface functionalization using a fluorescently labeled RGD peptide. Post functionalization, ~10,000 MDA‐MB‐231Br cells were seeded on top of the hydrogels and cultured for 5 days. We found that an increase in RGD concentration led to changes in cell morphology, with cells transitioning from a rounded to spindle‐like morphology as well as an increase in cell spreading area. Also, an increase in RGD concentration resulted in an increase in cell proliferation. Cellular dormancy was assessed using the ratio of phosphorylated extracellular signal‐regulated kinase 1/2 (p‐ERK) to phosphorylated p38 (p‐p38) positivity, which was significantly lower in hydrogels without RGD and in hydrogels with lowest RGD concentration compared to hydrogels functionalized with higher RGD concentration. We also demonstrated that the HA hydrogel‐induced cellular dormancy was reversible. Finally, we demonstrated the involvement of β1 integrin in mediating cell phenotype in our hydrogel platform. Overall, our results provide insight into the role of biochemical cues in regulating dormancy versus proliferation in brain metastatic breast cancer cells.more » « less
-
Abstract Human chondrocytes are responsible for cartilage repair and homeostasis through metabolic production of precursors to collagen and other matrix components. This metabolism is sensitive both to the availability of media energy sources as well as the local temperature. Central carbon metabolites such as glucose and glutamine are essential not only for producing energetic compounds such as ATP and NADH, but also for assembling collagen and aggrecan from non-essential amino acid precursors. The rate at which this metabolism takes place directly relates to temperature: a moderate increase in temperature results in faster enzyme kinetics and faster metabolic processes. Furthermore, these biological processes are exothermic and will generate heat as a byproduct, further heating the local environment of the cell. Prior studies suggest that mechanical stimuli affect levels of central metabolites in three-dimensionally cultured articular chondrocytes. But these prior studies have not determined if articular chondrocytes produce measurable heat. Thus,the goal of this studyis to determine if three-dimensionally encapsulated chondrocytes are capable of heat production which will improve our knowledge of chondrocyte central metabolism and further validate in vitro methods. Here we show the results of microcalorimetric measurements of heat generated by chondrocytes suspended in agarose hydrogels over a 2-day period in PBS, glucose, and glutamine media. The results show that a significant amount of heat is generated by cells (Cells Only: 3.033 ± 0.574 µJ/cell, Glucose: 2.791 ± 0.819 µJ/cell, Glutamine: 1.900 ± 0.650 µJ/cell) versus the absence of cells (No Cells: 0.374 ± 0.251 µJ/cell). This suggests that cells which have access to carbon sources in the media or as intracellular reserves will generate a significant amount of heat as they process these metabolites, produce cellular energy, and synthesize collagen precursors. The length of the microcalorimeter experiment (48 h) also suggests that the metabolism of articular chondrocytes is slower than many other cells, such as human melanoma cells, which can produce similar quantities of heat in less than an hour. These data broadly suggest that chondrocyte metabolism is sensitive to the available nutrients and has the potential to alter cartilage temperature through metabolic activity.more » « less
