CD206+ tendon resident macrophages and their potential crosstalk with fibroblasts and the ECM during tendon growth and maturation.

Resident macrophages exist in a variety of tissues, including tendon, and play context-specific roles in their tissue of residence. In this study, we define the spatiotemporal distribution and phenotypic profile of tendon resident macrophages and their crosstalk with neighboring tendon fibroblasts and the extracellular matrix (ECM) during murine tendon development, growth, and homeostasis. Fluorescent imaging of cryosections revealed that F4/80+ tendon resident macrophages reside adjacent to Col1a1-CFP+ Scx-GFP+ fibroblasts within the tendon fascicle from embryonic development (E15.5) into adulthood (P56). Through flow cytometry and qPCR, we found that these tendon resident macrophages express several well-known macrophage markers, including Adgre1 (F4/80), Mrc1 (CD206), Lyve1, and Folr2, but not Ly-6C, and express the Csf1r-EGFP ("MacGreen") reporter. The proportion of Csf1r-EGFP+ resident macrophages in relation to the total cell number increases markedly during early postnatal growth, while the density of macrophages per mm2 remains constant during this same time frame. Interestingly, proliferation of resident macrophages is higher than adjacent fibroblasts, which likely contributes to this increase in macrophage proportion. The expression profile of tendon resident macrophages also changes with age, with increased pro-inflammatory and anti-inflammatory cytokine expression in P56 compared to P14 macrophages. In addition, the expression profile of limb tendon resident macrophages diverges from that of tail tendon resident macrophages, suggesting differential phenotypes across anatomically and functionally different tendons. As macrophages are known to communicate with adjacent fibroblasts in other tissues, we conducted ligand-receptor analysis and found potential two-way signaling between tendon fibroblasts and resident macrophages. Tendon fibroblasts express high levels of Csf1, which encodes macrophage colony stimulating factor (M-CSF) that acts on the CSF1 receptor (CSF1R) on macrophages. Importantly, Csf1r-expressing resident macrophages preferentially localize to Csf1-expressing fibroblasts, supporting the "nurturing scaffold" model for tendon macrophage patterning. Lastly, we found that tendon resident macrophages express high levels of ECM-related genes, including Mrc1 (mannose receptor), Lyve1 (hyaluronan receptor), Lair1 (type I collagen receptor), Ctss (elastase), and Mmp13 (collagenase), and internalize DQ Collagen in explant cultures. Overall, our study provides insights into the potential roles of tendon resident macrophages in regulating fibroblast phenotype and the ECM during tendon growth.

Csf1 from marrow adipogenic precursors is required for osteoclast formation and hematopoiesis in bone.

Colony-stimulating factor 1 (Csf1) is an essential growth factor for osteoclast progenitors and an important regulator for bone resorption. It remains elusive which mesenchymal cells synthesize Csf1 to stimulate osteoclastogenesis. We recently identified a novel mesenchymal cell population, marrow adipogenic lineage precursors (MALPs), in bone. Compared to other mesenchymal subpopulations, MALPs expressed Csf1 at a much higher level and this expression was further increased during aging. To investigate its role, we constructed MALP-deficient Csf1 CKO mice using AdipoqCre. These mice had increased femoral trabecular bone mass, but their cortical bone appeared normal. In comparison, depletion of Csf1 in the entire mesenchymal lineage using Prrx1Cre led to a more striking high bone mass phenotype, suggesting that additional mesenchymal subpopulations secrete Csf1. TRAP staining revealed diminished osteoclasts in the femoral secondary spongiosa region of Csf1 CKOAdipoq mice, but not at the chondral-osseous junction nor at the endosteal surface of cortical bone. Moreover, Csf1 CKOAdipoq mice were resistant to LPS-induced calvarial osteolysis. Bone marrow cellularity, hematopoietic progenitors, and macrophages were also reduced in these mice. Taken together, our studies demonstrate that MALPs synthesize Csf1 to control bone remodeling and hematopoiesis.

A brief history of tendon and ligament bioreactors: Impact and future prospects.

Bioreactors are powerful tools with the potential to model tissue development and disease in vitro. For nearly four decades, bioreactors have been used to create tendon and ligament tissue-engineered constructs in order to define basic mechanisms of cell function, extracellular matrix deposition, tissue organization, injury, and tissue remodeling. This review provides a historical perspective of tendon and ligament bioreactors and their contributions to this advancing field. First, we demonstrate the need for bioreactors to improve understanding of tendon and ligament function and dysfunction. Next, we detail the history and evolution of bioreactor development and design from simple stretching of explants to fabrication and stimulation of two- and three-dimensional constructs. Then, we demonstrate how research using tendon and ligament bioreactors has led to pivotal basic science and tissue-engineering discoveries. Finally, we provide guidance for new basic, applied, and clinical research utilizing these valuable systems, recognizing that fundamental knowledge of cell-cell and cell-matrix interactions combined with appropriate mechanical and chemical stimulation of constructs could ultimately lead to functional tendon and ligament repairs in the coming decades.

Decellularization and Recellularization of Cartilage.

Decellularization of cartilage enables the use of cartilage allografts or xenografts as natural scaffolds for repair and regeneration of injured cartilage. The preservation of the extracellular matrix ultrastructure of the graft makes this a promising tool for cartilage tissue engineering. We have optimized the decellularization protocol by enzymatically digesting proteoglycans while preserving the native collagen architecture. Here we describe our methods for cartilage decellularization and cell labeling for the tracking of infiltration for recellularization in detail.

Effects of Chondroitinase ABC-Mediated Proteoglycan Digestion on Decellularization and Recellularization of Articular Cartilage.

Articular cartilage has a limited capacity to heal itself and thus focal defects often result in the development of osteoarthritis. Current cartilage tissue engineering strategies seek to regenerate injured tissue by creating scaffolds that aim to mimic the unique structure and composition of native articular cartilage. Decellularization is a novel strategy that aims to preserve the bioactive factors and 3D biophysical environment of the native extracellular matrix while removing potentially immunogenic factors. The purpose of this study was to develop a procedure that can enable decellularization and recellularization of intact articular cartilage matrix. Full-thickness porcine articular cartilage plugs were decellularized with a series of freeze-thaw cycles and 0.1% (w/v) sodium dodecyl sulfate detergent cycles. Chondroitinase ABC (ChABC) was applied before the detergent cycles to digest glycosaminoglycans in order to enhance donor chondrocyte removal and seeded cell migration. Porcine synovium-derived mesenchymal stem cells were seeded onto the decellularized cartilage scaffolds and cultured for up to 28 days. The optimized decellularization protocol removed 94% of native DNA per sample wet weight, while collagen content and alignment were preserved. Glycosaminoglycan depletion prior to the detergent cycles increased removal of nuclear material. Seeded cells infiltrated up to 100 µm into the cartilage deep zone after 28 days in culture. ChABC treatment enhances decellularization of the relatively dense, impermeable articular cartilage by reducing glycosaminoglycan content. ChABC treatment did not appear to affect cell migration during recellularization under static, in vitro culture, highlighting the need for more dynamic seeding methods.