Vertebral body versus iliac crest bone marrow as a source of multipotential stromal cells: Comparison of processing techniques, tri-lineage differentiation and application on a scaffold for spine fusion

The potential use of bone progenitors, multipotential stromal cells (MSCs) helping spine fusion is increasing, but convenient MSC sources and effective processing methods are critical factors yet to be optimised. The aim of this study was to test the effect of bone marrow processing on the MSC abundance and to compare the differentiation capabilities of vertebral body-bone marrow (VB-BM) MSCs versus iliac crest-bone marrow (IC-BM) MSCs. We assessed the effect of the red blood cell lysis (ammonium chloride, AC) and density-gradient centrifugation (Lymphoprep™, LMP), on the extracted VB-BM and IC-BM MSC numbers. The MSC abundance (indicated by colony counts and CD45lowCD271high cell numbers), phenotype, proliferation and tri-lineage differentiation of VB-BM MSCs were compared with donor-matched IC-BM MSCs. Importantly, the MSC attachment and osteogenesis were examined when VB-BM and IC-BM samples were loaded on a beta-tricalcium phosphate scaffold. In contrast to LMP, using AC yielded more colonies from IC-BM and VB-BM aspirates (p = 0.0019 & p = 0.0201 respectively). For IC-BM and VB-BM, the colony counts and CD45lowCD271high cell numbers were comparable (p = 0.5186, p = 0.2640 respectively). Furthermore, cultured VB-BM MSCs exhibited the same phenotype, proliferative and adipogenic potential, but a higher osteogenic and chondrogenic capabilities than IC-BM MSCs (p = 0.0010 and p = 0.0005 for calcium and glycosaminoglycan (GAG) levels, respectively). The gene expression data confirmed higher chondrogenesis for VB-BM MSCs than IC-BM MSCs, but osteogenic gene expression levels were comparable. When loaded on Vitoss™, both MSCs showed a similar degree of attachment and survival, but a better osteogenic ability was detected for VB-BM MSCs as measured by alkaline phosphatase activity (p = 0.0386). Collectively, the BM processing using AC had more MSC yield than using LMP. VB-BM MSCs have a comparable phenotype and proliferative capacity, but higher chondrogenesis and osteogenesis with or without using scaffold than donor-matched IC-BM MSCs. Given better accessibility, VB-BM could be an ideal MSC source for spinal bone fusion

Evidence that tissue resident human enthesis γδ T-cells can produce IL-17A independently of IL-23R transcript expression

Objectives: Murine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of Î 3Î T-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated Î 3Î T-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis. Methods: Human spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal Î 3Î T-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from Î 3Î T-cell subsets and analysed by qPCR. Entheseal Î 3Î T-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR. Results: Entheseal Î 3Î T-cells were confirmed immunohistochemically with VÎ 1 and VÎ 2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal VÎ 2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the VÎ 1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both VÎ 1 and VÎ 2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the VÎ 1 subset following IL-23 stimulation. Conclusion: Spinal entheseal VÎ 1 and VÎ 2 subsets are tissue resident cells with inducible IL-17A production with evidence that the VÎ 1 subset does so independently of IL-23R expression

Defective proliferation and osteogenic potential with altered immunoregulatory phenotype of native bone marrow-multipotential stromal cells in atrophic fracture non-union

Bone marrow-Multipotential stromal cells (BM-MSCs) are increasingly used to treat complicated fracture healing e.g., non-union. Though, the quality of these autologous cells is not well characterized. We aimed to evaluate bone healing-related capacities of non-union BM-MSCs. Iliac crest-BM was aspirated from long-bone fracture patients with normal healing (U) or non-united (NU). Uncultured (native) CD271highCD45low cells or passage-zero cultured BM-MSCs were analyzed for gene expression levels, and functional assays were conducted using culture-expanded BM-MSCs. Blood samples were analyzed for serum cytokine levels. Uncultured NU-CD271highCD45low cells significantly expressed fewer transcripts of growth factor receptors, EGFR, FGFR1, and FGRF2 than U cells. Significant fewer transcripts of alkaline phosphatase (ALPL), osteocalcin (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were detected in NU-CD271highCD45low cells. Additionally, immunoregulation-related markers were differentially expressed between NU- and U-CD271highCD45low cells. Interestingly, passage-zero NU BM-MSCs showed low expression of immunosuppressive mediators. However, culture-expanded NU and U BM-MSCs exhibited comparable proliferation, osteogenesis, and immunosuppression. Serum cytokine levels were found similar for NU and U groups. Collectively, native NU-BM-MSCs seemed to have low proliferative and osteogenic capacities; therefore, enhancing their quality should be considered for regenerative therapies. Further research on distorted immunoregulatory molecules expression in BM-MSCs could potentially benefit the prediction of complicated fracture healing

Innate Lymphoid Cells are Present at Normal Human Enthesis Providing a Potential Mechanism for Spondyloarthropathy Pathogenesis

Background and objectives The pathogenesis of murine spondyloarthropathy (SpA) has been intimately linked to the presence of IL-23 responsive, innate like lymphocytes at peripheral and spinal enthesis. Human SpAs are associated with SNPs in genes related to the IL-23 pathway and drugs that block IL-12/23 have shown efficacy. We hypothesised that the normal human enthesis has a population of resident innate lymphoid cells (ILCs) that could be key in governing entheseal immune homeostasis partly via interaction with resident mesenchymal stromal cells (MSCs). Materials and methods Normal spinal enthesis were harvested from patients undergoing spinal decompression surgery and enzymatically digested prior to sorting or flow cytometry. Immunophenotyping and cell sorting was performed on enthesis samples harvested from 6 patients and unmatched peripheral blood. The expression of RORγt and key immunomodulatory transcripts was tested in sorted populations by RTqPCR. Anterior cruciate ligament and Achilles enthesis were obtained from patients with knee OA and Achilles tendon rupture and analysed by immunohistochemistry (IHC). Adherent cells from entheseal digest were cultured under standard MSC culture conditions and expression of known MSC markers was assessed by flow cytometry. Results All sorted samples contained ILC3s, median proportion 0.09% (range 0.015-0.63). Transcript analysis confirmed the expression of RORγt transcript in sorted ILC3 populations. ILC3s expressed 51-fold greater relative expression of RORγt in comparison to unsorted mononuclear cells. 5 of 6 sorted samples contained ILC2s, median proportion 0.20% (range 0-0.49). RORγt expression was detected in knee OA and there was widespread expression of RORγt in inflammatory infiltrates in injured enthesis as shown by IHC. Culture expanded adherent cells grew in characteristic fibroblastoid colonies and expressed phenotypic markers consistent with bone marrow derived MSCs. Conclusions Our findings show that both ILCs and MSCs are present in the normal human spinal enthesis. ILCs may also be greatly increased in frequency following injury. The co-localisation of ILC and MSC populations at the enthesis suggests a potential link between cellular dysregulation of the IL-23/17 axis and human SpA pathology