Neural and Physical Niches Orchestrate Skeletal Remodelling and Promote Cartilage Regeneration
The skeleton is densely innervated by sympathetic noradrenergic and cholinergic nerve fibres. While several studies suggest the bone-catabolic nature of noradrenergic innervation in decreasing osteoblast activity and promoting osteoclastogenesis, there is little known on the development and function of skeletal cholinergic innervation. Therefore, the first goal of my dissertation research was to elucidate potential secreted factors that promote cholinergic differentiation during postnatal skeletal development, and to examine the function of the cholinergic system in bone using genetic and pharmacological depletion of cholinergic neurons. Treatment of superior cervical ganglion cultures with interleukin-6 (IL-6) for 14 days induced cholinergic, and reduced noradrenergic, gene and protein expression. In vivo studies showed IL-6 expression in developing skeletal muscle cells, and depletion of IL-6 using targeted antibodies and IL-6-deficient mice led to reduced cholinergic innervation in developing long bones. Further, genetic ablation of GDNF family receptor-α2 (Gfra2) induced expression of tumour necrosis factor-alpha converting enzyme (TACE) in neurons, which promotes cleavage of IL-6 receptor leading to enhanced trans-IL-6 signalling, reducing cholinergic trans-differentiation. Therefore, during postnatal life, sprouting sympathetic neurons contact the periosteum and respond to IL-6 secreted by skeletal muscle, resulting in cholinergic differentiation when intrinsic TACE levels are low. Genetic lineage tracing of choline acetyltransferase (ChAT) confirmed dense cholinergic innervation in periosteum and cortical bone, with sparse branches reaching trabecular bone, and labelled bone-lining osteoprogenitors shown to amplify the cholinergic signal to the bone marrow parenchyma. Selective depletion of cholinergic neurons using Gfra2 knockout (Gfra2-/-) mice reduced cortical and trabecular bone mass, bone strength, and bone formation rate. While osteoclast numbers were unaffected in Gfra2-/- mice, osteocytes showed reduced dendritic connectivity and survival. Mechanistically, cholinergic fibres provide a rich source of the neurotrophic factor, Neurturin (NRTN), which supports osteocyte survival and growth. Loss of cholinergic innervation also led to increased expression of the Wnt inhibitor, sclerostin, in osteocytes, and inhibition of sclerostin in Gfra2-/- mice rescued deficits in bone formation. Overall, skeletal cholinergic innervation provides neurotrophic signals and suppresses sclerostin in osteocytes, promoting bone formation and osteocyte survival. Differentiated chondrocytes from human bone marrow stromal cells including skeletal stem cells (hBMSCs/SSCs) and induced pluripotent stem cells (hiPSCs) have the potential to permanently restore damaged cartilage in arthritic joints, yet chondrocyte hypertrophy is a major barrier for translational therapy. With chondrogenic differentiation, hBMSCs/SSCs undergo hypertrophy in vitro and mineralisation in vivo, leading to inferior fibrocartilage and bone formation. However, hBMSCs/SSCs attached to a fibrin microbead scaffold coated with hyaluronic acid (HyA-FMBs) produce hyaline-like cartilage for up to 28 weeks in vivo. Therefore, the second goal of my dissertation research was to examine the signalling pathways that govern the development of hypertrophic-resistant chondrocytes using the HyA-FMB model system, and to guide hiPSCs to stable chondrocyte-like cells using this mechanistic knowledge. After one day of chondrogenic differentiation in vitro, hBMSCs/SSCs attached to HyA-FMBs exhibited higher expression of extracellular matrix proteins—including Insulin-like Growth Factor Binding Protein-5 (IGFBP5) and Matrix Gla Protein (MGP)—and decreased Bone Morphogenic Protein (BMP) signalling evidenced by pathway analysis. However, BMP signalling was restored by day 5, and increased by day 10, in a chondrogenic subpopulation enriched for IGFBP5 and MGP, accompanied by diminished expression of hypertrophic and osteogenic markers (COL10A1, ALPL, IBSP, and SPP1) exclusively in hBMSCs/SSCs attached to HyA-FMBs. Transcriptomic measurements confirmed increased BMP signalling in stable hyaline-like cartilage produced by ectopic transplantation of hBMSCs/SSCs attached to HyA-FMBs. Using this knowledge, I developed a serum-free hiPSC differentiation strategy that inhibited, then activated BMP signalling in a purified SOX9+ subpopulation that naturally detaches from monolayer cultures (termed “chondrospheroids”). Treatment of SOX9+ chondrospheroids with BMP-2 and GDF-5 produced uniform and stable expression of COL2A1, ACAN, and PRG4 and minimal expression of COL10A1 in vitro and in vivo. Osteochondral transplantation of day 35 chondrospheroids, which mimicked the transcriptional identity of a foetal chondrocyte, produced stable hyaline-like cartilage for up to 5 months in NSG mice and SRG rats when attached to HyA-FMBs. Therefore, BMP signalling activates and maintains a hypertrophic-resistant chondrogenic cell enriched for SOX9, MGP and IGFBP5 during chondrogenic differentiation.
bone marrow stromal cell, bone remodelling, cholinergic, chondrogenesis, fibrin microbead, induced pluripotent stem cell, osteocyte, skeletal stem cell, sympathetic
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