Treatment of disorders that disrupt homeostasis of skeletal tissues, such as osteoarthritis, and developmental skeletal diseases, such as overgrowth syndromes, requires a precise understanding of skeletal development. Osteoarthritis is a whole joint disorder that can be caused by articular cartilage defects that illicit an inflammatory immune response. Articular cartilage does not have intrinsic processes for repair or regeneration, so it is necessary to develop tissue constructs to repair the native tissue.

Since articular cartilage is not capable of repair, it is difficult to engraft cartilage tissue constructs. As such, it is necessary to develop a layered osteochondral construct that can integrate with the bone layer just beneath the articular cartilage. Generation of a layered tissue construct presents a major challenge because bone and cartilage differentiation require very different stimulating cues. Here, we attempted to develop a multi-compartmental “bi-culture” system to promote spatially restricted differentiation of an osteochondral tissue construct. We use a progenitor cell derived from articular cartilage called a cartilage progenitor cell which is capable of differentiating into articular-like chondrocytes, hypertrophic-like chondrocytes, or osteoblasts, all of which play important roles in osteochondral tissue development. We use a demineralized human cancellous bone scaffold which has demonstrated its propensity to support bone and cartilage formation and success in engrafting with the host tissues. However, we find that the osteogenic and chondrogenic cues we use to develop the osteochondral tissue construct are insufficient, and we need to apply more native-like stimulation of tissue development to obtain an osteochondral construct.

Epigenetic regulation has steadily gained attention in the field of orthopaedics. It has become clear that a variety of epigenetic factors play important roles in the development and homeostasis of skeletal tissues. Among these epigenetic factors are microRNAs. MicroRNA profiling studies have identified many differentially expressed miRNAs in chondrocytes at distinct sites of developing limb growth plate. Among the top differentially expressed microRNAs are miR181a-1 and miR-138.

miR-181a-1 is part of the miR-181a/b-1 cluster and has a reported pro-differentiation function in multiple in vitro differentiation assays. Here, we used lentiviral approaches to over-express this microRNA cluster during chondrogenesis of cartilage progenitor cells in micromass pellet cultures and report a pro-differentiation role. Using bulk RNA-sequencing, we identify a number of pro-anabolic genes and pathways upregulated by miR-181a/b-1 over-expression during cartilage progenitor cell chondrogenesis. The gene most downregulated by miR-181a/b-1 overexpression was aquaporin-9 which has not been studied extensively in cartilage development or homeostasis. We report that aquaporin-9 is expressed intracellularly during cartilage progenitor cell chondrogenesis and that it is not a direct target of miR-181a/b-1. The miR-181a/b-1 cluster appears to positively regulate chondrogenesis and chondrocyte anabolism, and one of the mechanisms for this effect may be indirect downregulation of aquaporin-9.

miR-138 has been identified as an anti-proliferation, anti-adhesion, anti-motility agent, primarily in the context of cancers. Here, we used lentiviral approaches to over-express miR-138 during cartilage progenitor cell micromass pellet chondrogenesis and report that it has limited impact on differentiation. Bulk RNA-sequencing analysis detected only one significantly differentially expressed gene and minimal perturbation of chondrogenic-related signaling pathways. Our report adds to the ambiguous literature on the role of miR-138 in chondrogenesis.

Another class of epigenetic regulators that are known to regulate skeletal development is DNA methyltransferases. Mutations in one of member of this class, DNMT3A, are responsible for a skeletal overgrowth syndrome called Tatton-Brown-Rahman syndrome. Interestingly, TattonBrown-Rahman syndrome patients also have intellectual and behavioral disorders, so it falls under the umbrella of overgrowth and intellectual disability syndromes.

Recently, two different mouse models were generated with two distinct mutations in Dnmt3a, both of which are homologous to mutations found in human Tatton-Brown-Rahman syndrome patients. Here, we perform a thorough skeletal characterization on mice with either of the Dnmt3a mutations to assess the potential of these mice as an animal model for Tatton-BrownRahman syndrome. We report that these mice mimic some of the skeletal overgrowth phenotypes found in patients with these mutations. Investigating the cellular mechanism responsible for this overgrowth, we find that the growth plates in both Dnmt3a mutant mice are significantly thicker than their wild-type littermates during skeletal development. These findings indicate that both Dnmt3a mutant mice are good animal models for Tatton-Brown-Rahman syndrome.

Given that DNMT3A has been reported to have a pro-osteogenic and anti-osteoclastogenic function, we hypothesized that there may be additional skeletal phenotypes present in the Dnmt3a mutant mice. We expanded our skeletal characterization to include other skeletal parameters such as trabecular and cortical bone indices, bone mechanical properties, and bone marrow adipose tissue accumulation. We found reduced cortical bone thickness and impaired bone mechanical properties in both of the Dnmt3a mutants. We investigated osteoblast function and osteoclast number in the mice during development but did not identify a clear cellular mechanism. We also report a sex-specific and mutation-specific upregulation of bone marrow adipose tissue in mice with one of the Dnmt3a mutations. These additional skeletal phenotypes represent opportunity to improve our understanding of Tatton-Brown-Rahman syndrome and, more broadly, overgrowth and intellectual disability syndromes.

These investigations of microRNAs and DNA methyltransferases during in vitro chondrogenesis and in vivo skeletal overgrowth add to the rapidly expanding body of literature on epigenetic regulators in skeletal development. As we learn more about the functional roles of epigenetic regulators, we can better use them as tools to treat diseases and improve human health.