In Vitro Osteogenesis Study of Shell Nacre Cement with Older and Young Donor Bone Marrow Mesenchymal Stem/Stromal Cells.

Bone void-filling cements are one of the preferred materials for managing irregular bone voids, particularly in the geriatric population who undergo many orthopedic surgeries. However, bone marrow mesenchymal stem/stromal cells (BM-MSCs) of older-age donors often exhibit reduced osteogenic capacity. Hence, it is crucial to evaluate candidate bone substitute materials with BM-MSCs from the geriatric population to determine the true osteogenic potential, thus simulating the clinical situation. With this concept, we investigated the osteogenic potential of shell nacre cement (SNC), a bone void-filling cement based on shell nacre powder and ladder-structured siloxane methacrylate, using older donor BM-MSCs (age > 55 years) and young donor BM-MSCs (age < 30 years). Direct and indirect cytotoxicity studies conducted with human BM-MSCs confirmed the non-cytotoxic nature of SNC. The standard colony-forming unit-fibroblast (CFU-F) assay and population doubling (PD) time assays revealed a significant reduction in the proliferation potential (p < 0.0001, p < 0.05) in older donor BM-MSCs compared to young donor BM-MSCs. Correspondingly, older donor BM-MSCs contained higher proportions of senescent, ß-galactosidase (SA-ß gal)-positive cells (nearly 2-fold, p < 0.001). In contrast, the proliferation capacity of older donor BM-MSCs, measured as the area density of CellTrackerTM green positive cells, was similar to that of young donor BM-MSCs following a 7-day culture on SNC. Furthermore, after 14 days of osteoinduction on SNC, scanning electron microscopy with energy-dispersive spectroscopy (SEM-EDS) showed that the amount of calcium and phosphorus deposited by young and older donor BM-MSCs on SNC was comparable. A similar trend was observed in the expression of the osteogenesis-related genes BMP2, RUNX2, ALP, COL1A1, OMD and SPARC. Overall, the results of this study indicated that SNC would be a promising candidate for managing bone voids in all age groups.

Soluble low affinity nerve growth factor receptor (sLNGFR) may regulate pain in knee osteoarthritis.

OBJECTIVES: Nerve growth factor ß (ß-NGF) is a protein which is important to the development of neurons particularly those involved in the transmission of pain and is central to the experience of pain in osteoarthritis (OA). Direct NGF antagonism has been shown to reduce OA pain but is associated with rapidly progressive OA. The aim of the study is to investigate the ability of soluble neurotrophin receptors in the NGF pathway to modulate pain in OA. METHODS: Synovial fluid (SF) was obtained from the knee joints of 43 subjects who underwent total knee arthroplasty. Visual analogue scale (VAS) pain scores were obtained prior to surgery. Customised-automated-ELISAs and commercial-ELISAs and LEGENDplex™ were used to measure soluble low-affinity nerve growth factor (LNGFR), soluble tropomyosin receptor kinase (TrkA), proNGF, ß-NGF, other neurotrophins (NT) and cytokines including inflammatory marker TNF-α. RESULTS: The VAS score positively correlated with ß-NGF (r=0.34) and there was positive association trend with neurotrophin-3 (NT-3), BDNF and negative association trend with ProNGF. sLNGFR positively correlated with VAS (r=0.33). The ß-NGF/soluble TrkA ratio showed a strong positive correlation with VAS (r=0.80). In contrast, there was no correlation between pain and the ß-NGF/sLNGFR ratio (r=-0.08). TNF-α positively correlated with ß-NGF (r=0.83), NT-3 (r=0.66), and brain-derived neurotrophic factor (BDNF) (r=0.50) and negatively with ProNGF (r= -0.74) and positively correlated with both soluble TrkA (r=0.62), sLNGFR (r=0.26). CONCLUSIONS: This study suggests that endogenous or cleaved sLNGFR, but not soluble TrkA may participate in OA pain modulation thus supporting further research into soluble LNGFR as a therapeutic target in OA.

Cytokine receptor IL27RA is an NF-κB-responsive gene involved in CD38 upregulation in multiple myeloma.

Multiple myeloma (MM) shows constitutive activation of canonical and noncanonical nuclear factor κB (NF-κB) signaling via genetic mutations or tumor microenvironment (TME) stimulations. A subset of MM cell lines showed dependency for cell growth and survival on the canonical NF-κB transcription factor RELA alone, suggesting a critical role for a RELA-mediated biological program in MM pathogenesis. Here, we determined the RELA-dependent transcriptional program in MM cell lines and found the expression of the cell surface molecules interleukin-27 receptor-α (IL-27Rα) and the adhesion molecule JAM2 to be responsive to RELA at the messenger RNA and protein levels. IL-27Rα and JAM2 were expressed on primary MM cells at higher levels than on healthy long-lived plasma cells (PCs) in the bone marrow. IL-27 activated STAT1, and to a lesser extent STAT3, in MM cell lines and in PCs generated from memory B cells in an IL-21-dependent in vitro PC differentiation assay. Concomitant activity of IL-21 and IL-27 enhanced differentiation into PCs and increased the cell-surface expression of the known STAT target gene CD38. In accordance, a subset of MM cell lines and primary MM cells cultured with IL-27 upregulated CD38 cell-surface expression, a finding with potential implications for enhancing the efficacy of CD38-directed monoclonal antibody therapies by increasing CD38 expression on tumor cells. The elevated expression of IL-27Rα and JAM2 on MM cells compared with that on healthy PCs may be exploited for the development of targeted therapeutic strategies that modulate the interaction of MM cells with the TME.

Induced Periosteum-Mimicking Membrane with Cell Barrier and Multipotential Stromal Cell (MSC) Homing Functionalities.

The current management of critical size bone defects (CSBDs) remains challenging and requires multiple surgeries. To reduce the number of surgeries, wrapping a biodegradable fibrous membrane around the defect to contain the graft and carry biological stimulants for repair is highly desirable. Poly(ε-caprolactone) (PCL) can be utilised to realise nonwoven fibrous barrier-like structures through free surface electrospinning (FSE). Human periosteum and induced membrane (IM) samples informed the development of an FSE membrane to support platelet lysate (PL) absorption, multipotential stromal cells (MSC) growth, and the prevention of cell migration. Although thinner than IM, periosteum presented a more mature vascular system with a significantly larger blood vessel diameter. The electrospun membrane (PCL3%-E) exhibited randomly configured nanoscale fibres that were successfully customised to introduce pores of increased diameter, without compromising tensile properties. Additional to the PL absorption and release capabilities needed for MSC attraction and growth, PCL3%-E also provided a favourable surface for the proliferation and alignment of periosteum- and bone marrow derived-MSCs, whilst possessing a barrier function to cell migration. These results demonstrate the development of a promising biodegradable barrier membrane enabling PL release and MSC colonisation, two key functionalities needed for the in situ formation of a transitional periosteum-like structure, enabling movement towards single-surgery CSBD reconstruction.

Colony Formation, Migratory, and Differentiation Characteristics of Multipotential Stromal Cells (MSCs) from "Clinically Accessible" Human Periosteum Compared to Donor-Matched Bone Marrow MSCs.

Periosteum is vital for fracture healing, as a highly vascular and multipotential stromal cell- (MSC-) rich tissue. During surgical bone reconstruction, small fragments of periosteum can be "clinically accessible," yet periosteum is currently not ultilised, unlike autologous bone marrow (BM) aspirate. This study is aimed at comparing human periosteum and donor-matched iliac crest BM MSC content and characterising MSCs in terms of colony formation, growth kinetics, phenotype, cell migration patterns, and trilineage differentiation capacity. "Clinically accessible" periosteum had an intact outer fibrous layer, containing CD271+ candidate MSCs located perivasculary; the inner cambium was rarely present. Following enzymatic release of cells, periosteum formed significantly smaller fibroblastic colonies compared to BM (6.1 mm2 vs. 15.5 mm2, n = 4, P = 0.0006). Periosteal colonies were more homogenous in size (range 2-30 mm2 vs. 2-54 mm2) and on average 2500-fold more frequent (2.0% vs. 0.0008%, n = 10, P = 0.004) relative to total viable cells. When expanded in vitro, similar growth rates up to passage 0 (P0) were seen (1.8 population doublings (PDs) per day (periosteum), 1.6 PDs per day (BM)); however, subsequently BM MSCs proliferated significantly slower by P4 (4.3 PDs per day (periosteum) vs. 9.3 PDs per day (BM), n = 9, P = 0.02). In early culture, periosteum cells were less migratory at slower speeds than BM cells. Both MSC types exhibited MSC phenotype and trilineage differentiation capacity; however, periosteum MSCs showed significantly lower (2.7-fold) adipogenic potential based on Nile red : DAPI ratios with reduced expression of adipogenesis-related transcripts PPAR-γ. Altogether, these data revealed that "clinically accessible" periosteal samples represent a consistently rich source of highly proliferative MSCs compared to donor-matched BM, which importantly show similar osteochondral capacity and lower adipogenic potential. Live cell tracking allowed determination of unique morphological and migration characteristics of periosteal MSCs that can be used for the development of novel bone graft substitutes to be preferentially repopulated by these cells.

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration.

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor-matched, clinical-, and control laboratory-standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU-F) assay was carried out on bone marrow aspirate using clinical-standard PP-loaded electrospun poly(ϵ-caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical-standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory-standard PP. It induced suboptimal MSC proliferation compared to laboratory-standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP-loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical-standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329-1338, 2019.

Bone Marrow-Harvesting Technique Influences Functional Heterogeneity of Mesenchymal Stem/Stromal Cells and Cartilage Regeneration.

BACKGROUND: Connective tissue progenitors (CTPs) from native bone marrow (BM) or their culture-expanded progeny, often referred to as mesenchymal stem/stromal cells, represents a promising strategy for treatment of cartilage injuries. But the cartilage regeneration capacity of these cells remains unpredictable because of cell heterogeneity. HYPOTHESIS: The harvest technique of BM may highly influence stem cell heterogeneity and, thus, cartilage formation because these cells have distinct spatial localization within BM from the same bone. STUDY DESIGN: Controlled laboratory study. METHODS: CTPs obtained from the femur of patients undergoing total hip replacement by 2 harvest techniques-BM aspiration and BM collection-after bone rasping were immunophenotyped by flow cytometry and evaluated for chondrogenic ability. The spatial localization of different CTP subsets in BM was verified by immunohistochemistry. RESULTS: Cells from the BM after rasping were significantly more chondrogenic than the donor-matched aspirate, whereas no notable difference in their osteogenic or adipogenic potential was observed. The authors then assessed whether distinct immunophenotypically defined CTP subsets were responsible for the different chondrogenic capacity. Cells directly isolated from BM after rasping contained a higher percentage (mean, 7.2-fold) of CD45-CD271+CD56+ CTPs as compared with BM aspirates. The presence of this subset in the harvested BM strongly correlated with chondrogenic ability, showing that CD271+CD56+ cells are enriched in chondroprogenitors. Furthermore, evaluation of these CTP subsets in BM revealed that CD271+CD56+ cells were localized in the bone-lining regions whereas CD271+CD56- cells were found in the perivascular regions. Since the iliac crest remains a frequent site of BM harvest for musculoskeletal regeneration, the authors also compared the spatial distribution of these subsets in trabeculae of femoral head and iliac crest and found CD271+CD56+ bone-lining cells in both tissues. CONCLUSION: Chondrogenically distinct CTP subsets have distinct spatial localization in BM; hence, the harvest technique of BM determines the efficiency of cartilage formation. CLINICAL RELEVANCE: The harvest technique of BM may be of major importance in determining the clinical success of BM mesenchymal stem/stromal cells in cartilage repair.

Use of platelet lysate for bone regeneration - are we ready for clinical translation?

Current techniques to improve bone regeneration following trauma or tumour resection involve the use of autograft bone or its substitutes supplemented with osteoinductive growth factors and/or osteogenic cells such as mesenchymal stem cells (MSCs). Although MSCs are most commonly grown in media containing fetal calf serum, human platelet lysate (PL) offers an effective alternative. Bone marrow - derived MSCs grown in PL-containing media display faster proliferation whilst maintaining good osteogenic differentiation capacity. Limited pre-clinical investigations using PL-expanded MSCs seeded onto osteoconductive scaffolds indicate good potential of such constructs to repair bone in vivo. In an alternative approach, nude PL-coated scaffolds without seeded MSCs have been proposed as novel regenerative medicine devices. Even though methods to coat scaffolds with PL vary, in vitro studies suggest that PL allows for MSC adhesion, migration and differentiation inside these scaffolds. Increased new bone formation and vascularisation in comparison to uncoated scaffolds have also been observed in vivo. This review outlines the state-of-the-art research in the field of PL for ex vivo MSC expansion and in vivo bone regeneration. To minimise inconsistency between the studies, further work is required towards standardisation of PL preparation in terms of the starting material, platelet concentration, leukocyte depletion, and the method of platelet lysis. PL quality control procedures and its "potency" assessment are urgently needed, which could include measurements of key growth and attachment factors important for MSC maintenance and differentiation. Furthermore, different PL formulations could be tailor-made for specific bone repair indications. Such measures would undoubtedly speed up clinical translation of PL-based treatments for bone regeneration.

Do skeletal muscle MSCs in humans contribute to bone repair? A systematic review.

Mesenchymal stem cells (MSC) from bone marrow and periosteum are known to be heavily involved in fracture repair and bone regeneration is thought to be impaired when the surrounding skeletal muscle is damaged. Recent literature from mouse in vivo models suggest that cells originating from skeletal muscle can occupy a fracture callus during open fracture repair when periosteum is compromised. This systematic review set out to ascertain whether there are MSCs residing in human skeletal muscle and whether cells from human skeletal muscle are capable of forming bone in vitro and in vivo. Original journal articles were selected if they included the terms "skeletal muscle" and "mesenchymal" and used human skeletal muscle samples. Between January 2005 and September 2016, 1000 articles were screened of which, 16 studies met the inclusion criteria for this review. Human skeletal muscle derived cells (SMDC) had the MSC phenotype, positive for CD73, CD90 and CD105 and negative for CD34 and CD45 as well as the potential to differentiate into osteoblasts, chondrocytes and adipocytes in vitro. In addition, SMDC could form bone in vivo when seeded onto an osteoinductive scaffold. A subset of SMDC expressing a pericyte marker (PDGFRα) also expressed the MSC phenotype and were more osteogenic in vivo in comparison to SMDC expressing a satellite cell marker (CD56). The studies included were limited through variation of SMDC extraction methods and tissue culture conditions, which causes heterogeneuous cell cultures. Also, in vitro differentiation assays were not always carried out with bone marrow MSC positive controls. Current evidence suggests that cells with the MSC phenotype reside within human skeletal muscle and are capable of in vivo bone formation in combination with osteoinductive bone scaffolds. This has implications of future development of guided bone regeneration strategies to enhance large bone defect repair, whereby more thought into whether the fracture site should be "blocked" from the skeletal muscle should be carried out.