Extracellular matrix derived from chondrocytes promotes rapid expansion of human primary chondrocytes in vitro with reduced dedifferentiation.

A significant expansion of autologous chondrocytes in vitro is required for cell-based cartilage repair. However, the in vitro expansion of chondrocytes under standard culture conditions inevitably leads to the dedifferentiation of chondrocytes and contributes to suboptimal clinical outcomes. To address this challenge, we focused our efforts on developing an improved in vitro expansion protocol, which shortens the expansion time with decreased dedifferentiation. It is known that the tissue microenvironment plays a critical role in regulating the cellular functions of resident cells and provides guidance in tissue-specific regeneration. We hypothesized that chondrocyte extracellular matrix (ECM) mimics a native microenvironment and that it may support chondrocyte expansion in vitro. To test this hypothesis, we prepared decellularized ECMs from allogeneic human articular chondrocytes (HAC) (AC-ECM) and bone marrow stromal cells (BM-ECM) and studied their effects on the in vitro expansion of primary HAC. The differential composition and physical properties of these two ECMs were revealed by mass spectrometry and atomic force microscopy. Compared with standard tissue culture polystyrene (TCP) or BM-ECM, HAC cultured on AC-ECM proliferated faster and maintained the highest ratio of COL2A1/COL1A1. Furthermore, a pellet culture study demonstrated that cells expanded on AC-ECM produced a more cartilage-like ECM than cells expanded on BM-ECM or TCP. This is the first report on modulating chondrocyte expansion and dedifferentiation using cell type-specific ECM and on identifying AC-ECM as a preferred substrate for in vitro expansion of HAC cell-based therapies. STATEMENT OF SIGNIFICANCE: To reduce the dedifferentiation of chondrocytes during in vitro expansion, cell type-specific extracellular matrix (ECM), which mimics a native microenvironment, was prepared from human articular chondrocytes (AC-ECM) or bone marrow stromal cells (BM-ECM). As demonstrated by mass spectrometry and atomic force microscopy, AC-ECM and BM-ECM have differential ECM compositions and physical characteristics. Human articular chondrocytes (HAC) expanded faster and maintained a better chondrocyte phenotype on AC-ECM than on BM-ECM or a standard culture surface. AC-ECM has potential to be developed for expanding HAC for cell-based therapies.

The NLRP1 inflammasome attenuates colitis and colitis-associated tumorigenesis.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are a diverse family of pattern recognition receptors that are essential mediators of inflammation and host defense in the gastrointestinal system. Recent studies have identified a subgroup of inflammasome forming NLRs that modulate the mucosal immune response during inflammatory bowel disease (IBD) and colitis associated tumorigenesis. To better elucidate the contribution of NLR family members in IBD and cancer, we conducted a retrospective analysis of gene expression metadata from human patients. These data revealed that NLRP1, an inflammasome forming NLR, was significantly dysregulated in IBD and colon cancer. To better characterize the function of NLRP1 in disease pathogenesis, we used Nlrp1b(-/-) mice in colitis and colitis-associated cancer models. In this paper, we report that NLRP1 attenuates gastrointestinal inflammation and tumorigenesis. Nlrp1b(-/-) mice demonstrated significant increases in morbidity, inflammation, and tumorigenesis compared with wild-type animals. Similar to data previously reported for related inflammasome forming NLRs, the increased inflammation and tumor burden was correlated with attenuated levels of IL-1ß and IL-18. Further mechanistic studies using bone marrow reconstitution experiments revealed that the increased disease pathogenesis in the Nlrp1b(-/-) mice was associated with nonhematopoietic-derived cells and suggests that NLRP1 functions in the colon epithelial cell compartment to attenuate tumorigenesis. Taken together, these data identify NLRP1 as an essential mediator of the host immune response during IBD and cancer. These findings are consistent with a model whereby multiple NLR inflammasomes attenuate disease pathobiology through modulating IL-1ß and IL-18 levels in the colon.

Caspase-11 attenuates gastrointestinal inflammation and experimental colitis pathogenesis.

Nucleotide-binding domain and leucine-rich repeat containing protein inflammasome formation plays an essential role in modulating immune system homeostasis in the gut. Recently, a caspase-11 noncanonical inflammasome has been characterized and appears to modulate many biological functions that were previously considered to be solely dependent on caspase-1 and the canonical inflammasome. To better elucidate the function of this noncanonical inflammasome during inflammatory bowel disease, experimental colitis was induced in wild-type and Casp11(-/-) mice utilizing dextran sulfate sodium (DSS). Here, we report that caspase-11 attenuates acute experimental colitis pathogenesis. Casp11(-/-) mice showed significantly increased morbidity and colon inflammation following DSS exposure. Subsequent cytokine analysis revealed that IL-1ß and IL-18 levels in the colon were significantly reduced in the Casp11(-/-) mice compared with the wild-type animals. Additional mechanistic studies utilizing IL-1ß and IL-18 reconstitution revealed that Casp11(-/-) hypersensitivity was associated with the loss of both of these cytokines. Bone marrow reconstitution experiments further revealed that caspase-11 gene expression and function in both hematopoietic- and nonhematopoietic-derived cells contribute to disease attenuation. Interestingly, unlike caspase-1, caspase-11 does not appear to influence relapsing remitting disease progression or the development of colitis-associated tumorigenesis. Together, these data identify caspase-11 as a critical factor protecting the host during acute DSS-induced colonic injury and inflammation but not during chronic inflammation and tumorigenesis.