Identification of a novel, MSC-induced macrophage subtype via single-cell sequencing: implications for intervertebral disc degeneration therapy.

Intervertebral disc (IVD) degeneration is a common pathological condition associated with low back pain. Recent evidence suggests that mesenchymal signaling cells (MSCs) promote IVD regeneration, but underlying mechanisms remain poorly defined. One postulated mechanism is via modulation of macrophage phenotypes. In this manuscript, we tested the hypothesis that MSCs produce trophic factors that alter macrophage subsets. To this end, we collected conditioned medium from human, bone marrow-derived STRO3+ MSCs. We then cultured human bone marrow-derived macrophages in MSC conditioned medium (CM) and performed single cell RNA-sequencing. Comparative analyses between macrophages cultured in hypoxic and normoxic MSC CM showed large overlap between macrophage subsets; however, we identified a unique hypoxic MSC CM-induced macrophage cluster. To determine if factors from MSC CM simulated effects of the anti-inflammatory cytokine IL-4, we integrated the data from macrophages cultured in hypoxic MSC CM with and without IL-4 addition. Integration of these data sets showed considerable overlap, demonstrating that hypoxic MSC CM simulates the effects of IL-4. Interestingly, macrophages cultured in normoxic MSC CM in the absence of IL-4 did not significantly contribute to the unique cluster within our comparison analyses and showed differential TGF-ß signaling; thus, normoxic conditions did not approximate IL-4. In addition, TGF-ß neutralization partially limited the effects of MSC CM. In conclusion, our study identified a unique macrophage subset induced by MSCs within hypoxic conditions and supports that MSCs alter macrophage phenotypes through TGF-ß-dependent mechanisms.

Evaluation of Amniotic Multipotential Tissue Matrix to Augment Healing of Demineralized Bone Matrix in an Animal Calvarial Model.

Amniotic multipotential tissue matrix (AmnioMTM) is a membrane material derived from placental tissues and rich in growth factors that have been reported to have potential in healing bone. This study hypothesized that demineralized bone matrix (DBM) supplemented with AmnioMTM would accelerate healing and bone formation as compared with DBM alone in a critical size (10 mm) rat calvarial bone defect model. Five DBM grafts and 5 DBM supplemented with AmnioMTM grafts were implanted in a 10-mm critical sized defect in 10 rats (1 implant per rat). After 4 weeks, animals were euthanized and defects evaluated by microCT and histology. There were no statistical differences in microCT data for mineral density, percent bone fill, or bone surface to volume ratios between groups, though the bone surface to volume ratio for the amnio-supplemented group suggested increased osteoid activity as compared with the DBM alone group. Histological data also indicated active osteoid activity and induced bone formation in the center of defects implanted with AmnioMTM supplemented graft as compared with DBM graft alone suggesting some potential osteoinductive potential. However, there was no significant difference at the mean percent of newly mineralized bone in the DBM group defect as compared with the AmnioMTM supplemented graft material. These data suggest that while bone formation was not increased at this early time point, the increased osteoid activity and the induction of new bone in the middle of the defect by the AmnioMTM indicates that further study is needed to assess its potential benefit to bone healing and regeneration.

Fabrication of crosslinked carboxymethylchitosan microspheres and their incorporation into composite scaffolds for enhanced bone regeneration.

Carboxymethylchitosan (CMCS) microspheres were prepared by the carboxymethylation of chitosan (CS) beads using monochloroacetic acid. The CMCS microspheres were crosslinked using two different methods: the amine-amine crosslinker genipin and carbodiimide chemistry, yielding Gen-X CMCS and X-CMCS beads, respectively. The Gen-X CMCS beads were found to have poor degradation and drug release profiles. The X-CMCS microspheres displayed good potential for use in tissue engineering applications in which degradation and local drug delivery are desired. The X-CMCS beads displayed enzymatic degradation of 82.7 ± 1.2% in 100 µg/mL lysozyme after 1 month. An extended release of rhBMP-2 for at least 45 days was also observed with the X-CMCS microspheres. Scaffolds were formed by fusing beads together, and the X-CMCS beads were successfully incorporated into composite X-CMCS/CS scaffolds. The composite scaffolds had increased degradation of 14.5 ± 6.6% compared to 0.5 ± 0.4% for CS-only scaffolds, and the X-CMCS/CS scaffolds released more rhBMP-2 at all timepoints. The composite scaffolds also supported the attachment and proliferation of SAOS-2 cells. The addition of X-CMCS beads resulted in fabrication of scaffolds with improved properties for use in bone tissue engineering.

Physical properties and in vitro evaluation of collagen-chitosan-calcium phosphate microparticle-based scaffolds for bone tissue regeneration.

Due to limitations of bone autografts and allografts, synthetic bone grafts using osteoconductive biomaterials have been designed. In this study, collagen-chitosan-calcium phosphate microparticle-based scaffolds fused with glycolic acid were compared to their counterparts without collagen in terms of degradation, cytocompatibility, porosity, and Young's modulus. It was found that 26-30% collagen was incorporated and that hydroxyapatite was present. Moreover, there were no differences between control and collagen scaffolds in degradation, cytocompatibility, porosity, and Young's modulus. In general, scaffolds exhibited 23% porosity, 0.6-1.2 MPa Young's modulus, 23% degradation over 4 weeks, and supported a four to seven fold increase in osteoblast cell number over 7 days in culture. Collagen can be incorporated into these bone graft substitute scaffolds, which show an improved degradation profile.

Preparation and functional assessment of composite chitosan-nano-hydroxyapatite scaffolds for bone regeneration.

Composite chitosan-nano-hydroxyapatite microspheres and scaffolds prepared using a co-precipitation method have shown potential for use in bone regeneration. The goal of this research was to improve the functional properties of the composite scaffolds by modifying the fabrication parameters. The effects of degree of deacetylation (DDA), drying method, hydroxyapatite content and an acid wash on scaffold properties were investigated. Freeze-dried 61% DDA scaffolds degraded faster (3.5 ± 0.5% mass loss) than air-dried 61% DDA scaffolds and 80% DDA scaffolds, but had a lower compressive modulus of 0.12 ± 0.01 MPa. Air-dried 80% DDA scaffolds displayed the highest compressive modulus (3.79 ± 0.51 MPa) and these scaffolds were chosen as the best candidate for use in bone regeneration. Increasing the amount of hydroxyapatite in the air-dried 80% DDA scaffolds did not further increase the compressive modulus of the scaffolds. An acid wash procedure at pH 6.1 was found to increase the degradation of air-dried 80% DDA scaffolds from 1.3 ± 0.1% to 4.4 ± 0.4%. All of the formulations tested supported the proliferation of SAOS-2 cells.

Osteoinductivity Assessment of BMP-2 Loaded Composite Chitosan-Nano-Hydroxyapatite Scaffolds in a Rat Muscle Pouch.

The objective of this study was to evaluate the osteoinductivity of composite chitosan-nano-hydroxyapatite scaffolds in a rat muscle pouch model. Previous in vitro characterization demonstrated the ability of the scaffolds to promote bone regeneration and as a carrier for local delivery of BMP-2. Composite microspheres were prepared using a co-precipitation method, and scaffolds were fabricated using an acid wash to adhere beads together. To determine the in vivo osteoinductivity of the scaffolds, the following groups (n = 6) were implanted into muscle pouches created in the latissimus dorsi of Sprague Dawley rats: (A) lyophilized scaffolds without rhBMP-2, (B) lyophilized scaffolds with rhBMP-2, (C) non-lyophilized scaffolds with rhBMP-2, and (D) absorbable collagen sponge with rhBMP-2 (control). Groups B, C, and D were loaded with 4 mL of a 9.0 µg/mL solution of rhBMP-2 for 48 h. The rats were sacrificed after one month and samples were analyzed for amount of residual implant material, new bone, and osteoid. Although the experimental groups displayed minimal degradation after one month, all of the scaffolds contained small amounts of woven bone and considerable amounts of osteoid. Approximately thirty percent of the open space available for tissue ingrowth in the scaffolds contained new bone or osteoid in the process of mineralization. The ability of the composite scaffolds (with and without BMP-2) to promote ectopic bone growth in vivo was demonstrated.

Lyophilization to improve drug delivery for chitosan-calcium phosphate bone scaffold construct: a preliminary investigation.

Lyophilization was evaluated in chitosan-calcium phosphate microspheres and scaffolds to improve drug delivery of growth factors and antibiotics for orthopedic applications. The dual delivery of an antibiotic and a growth factor from a composite scaffold would be beneficial for treatment of complex fracture sites, such as comminuted fractures and segmental bone defects. The aim of this investigation was to increase the loading capacity of the composite by taking advantage of the increased porosity, due to lyophilization, and to produce an extended elution profile using a secondary chitosan-bead coating. The physiochemical properties of the composite were investigated, and loading and elution studies were performed with alkaline phosphatase (ALP), bone morphogenetic protein-2 (BMP-2), and amikacin. Lyophilization was found to increase the surface area of scaffolds by over 400% and the porosity of scaffolds by 50%. Using ALP as a model protein, the loading capacity was increased by lyophilization from 4.3 +/- 2.5 to 24.6 +/- 3.6 microg ALP/mg microspheres, and the elution profile was extended by a supplemental chitosan coating. The loading capacity of BMP-2 for composite microspheres was increased from 74.4 +/- 3.7 to 102.1 +/- 8.0 microg BMP-2/g microspheres with lyophilization compared with nonlyophilized microspheres. The elution profiles of BMP-2 and the antibiotic amikacin were not extended with the supplemental coating. Additional investigations are planned to improve these elution characteristics for growth factors and antibiotics.