Human Marrow-derived Mesenchymal Stromal Cells Research Articles

Polycaprolactone/β-Tricalcium Phosphate Composite Scaffolds with Advanced Pore Geometries Promote Human Mesenchymal Stromal Cells' Osteogenic Differentiation.

Critical-sized mandibular bone defects, arising from, for example, resections after tumor surgeries, are currently treated with autogenous bone grafts. This treatment is considered very invasive and is associated with limitations such as morbidity and graft resorption. Tissue engineering approaches propose to use 3D scaffolds that combine structural features, biomaterial properties, cells, and biomolecules to create biomimetic constructs. However, mimicking the complex anatomy and composition of the mandible poses a challenge in scaffold design. In our study, we evaluated the dual effect of complex pore geometry and material composition on the osteogenic potential of 3D printed scaffolds. The scaffolds were made of polycaprolactone (PCL) alone (TCP0), or with a high concentration of β-tricalcium phosphate (β-TCP) up to 40% w/w (TCP40), with two complex pore geometries, namely a star- (S) and a diamond-like (D) shape. Scanning electron microscopy and microcomputed tomography images confirmed high fidelity during the printing process. The D-scaffolds displayed higher compressive moduli than the corresponding S-scaffolds. TCP40 scaffolds in simulated body fluid showed deposition of minerals on the surface after 28 days. Subsequently, we assessed the differentiation of seeded bone marrow-derived human mesenchymal stromal cells (hMSCs) over 28 days. The early expression of RUNX2 in the cell nuclei confirmed the commitment toward an osteogenic phenotype. Moreover, alkaline phosphatase (ALP) activity and collagen deposition displayed an increasing trend in the D-scaffolds. Collagen type I was mainly present in the deposited extracellular matrix (ECM), confirming deposition of bone matrix. Finally, Alizarin Red staining showed successful mineralization on all the TCP40 samples, with higher values for the S-shaped scaffolds. Taken together, our study demonstrated that the complex pore architectures of scaffolds comprised TCP40 stimulated osteogenic differentiation and mineralization of hMSCs in vitro. Future research will aim to validate these findings in vivo.

Class I Biocompatible DLP-Printed Acrylate Impairs Adhesion and Proliferation of Human Mesenchymal Stromal Cells in Indirect Cytotoxicity Assay.

The popular method of digital light processing 3D printing (DLP) for complex and individual laboratory equipment requires materials that are as inert as possible for use in contact with cells for subsequent investigations. However, the per se incomplete curing of acrylate resins by UV light leaves residuals that are not suitable for cell culture application. Therefore, we evaluated the cytotoxicity of four commercially available acrylate resins with bone marrow-derived human mesenchymal stromal cells (BM-hMSC) in an indirect cytotoxicity test. This involved incubating the printed cylinders in Transwell™ inserts for 7 days. While the degree of crosslinking did not increase significantly between freshly printed and stored samples (3 weeks in ambient conditions), the storage improved the material's performance in terms of cytocompatibility. The DNA amount and LDH activity showed a direct influence of the resin residuals on cell adhesion. The class I acrylate Surgical Guide™ left no adherent cells after 7 days, regardless of previous storage. In comparison, the Basic Ivory™ resin after storage allowed same amount of adherent cells after 7 days as the polystyrene reference. We conclude that resin residuals of certain materials are released, which allows the use of the resins in indirect contact with cells thereafter.

Mesenchymal stromal cell mitochondrial transfer to human induced T-regulatory cells mediates FOXP3 stability

The key obstacle to clinical application of human inducible regulatory T cells (iTreg) as an adoptive cell therapy in autoimmune disorders is loss of FOXP3 expression in an inflammatory milieu. Here we report human iTreg co-cultured with bone marrow-derived mesenchymal stromal cells (MSCs) during short-term ex vivo expansion enhances the stability of iTreg FOXP3 expression and suppressive function in vitro and in vivo, and further that a key mechanism of action is MSC mitochondrial (mt) transfer via tunneling nanotubules (TNT). MSC mt transfer is driven by mitochondrial metabolic function (CD39/CD73 signaling) in proliferating iTreg and promotes iTreg expression of FOXP3 stabilizing factors BACH2 and SENP3. These results elucidate cellular and molecular mechanisms underlying human MSC mt transfer to proliferating cells. MSC mt transfer stabilizes FOXP3 expression in iTregs, thereby enhancing and sustaining their suppressive function in inflammatory conditions in vitro and in vivo.

Benefits of photopolymerizable hydrogels for the effective delivery of raav vectors in human bone marrow derived mesenchymal stem cells

Purpose: Injured adult articular cartilage has a limited ability for self-healing. Administration of genetically modified bone marrow-derived mesenchymal stromal cells (MSCs) in cartilage defects is a promising approach to enhance cartilage repair, especially when using clinically relevant recombinant adeno-associated virus (rAAV) vectors [1,2]. Yet, the presence of neutralizing antibodies against the viral capsid proteins in patients [3] may impair effective gene transfer in translational protocols. Scaffold-guided rAAV gene transfer has the potential to overcome such an issue by protecting the vectors from neutralization while increasing their gene transfer efficacy by controlling their spatial and temporal delivery [4-7]. Here, we examined the feasibility of delivering rAAV vectors in human MSCs (hMSCs) via photopolymerizable hydrogel systems [8,9] as future, effective platform for cartilage repair. Methods: rAAV vectors were packaged, purified, and titrated as previously described [10,11]. rAAV-lacZ carries the E. coli β-galactosidase (lacZ) reporter gene and rAAV-RFP the Discosoma sp. red fluorescent protein gene (RFP) [10,11]. Cy3 labeling of the rAAV vectors was performed using a Cy3 Ab Labeling Kit (Amersham/GE Healthcare) with detection by live fluorescence imaging as previously described [12]. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty (n = 2). hMSCs were prepared as previously described [10,11]. Cells (passage 1) were seeded in 48-well plates (5,000 cells/well), maintained in DMEM, 10% FBS, 100 U/ml penicillin G, 100 μl/ml streptomycin, and incubated at 37ºC during 12 h before addition of the various hydrogel systems. The hydrogel systems (FID119 F3 and FID119 F4) were prepared by overnight dissolution of the compounds (300 mg of FID119 F3, i.e. F3; 200 mg of FID119 F4, i.e. F4) 10 ml NaCl 0.9% (w/v). The F3 and F4 hydrogel systems were mixed with rAAV-lacZ or rAAV-RFP in equal parts using a BTC® Medical Europe UV-Lamp (5 min) and directly applied to the cultures for up to 21 days. Release of rAAV from the hydrogels was evaluated by AAV Titration ELISA (Progen) in conditioned culture medium using a permeable support system as previously described [12]. Transgene (RFP) expression was detected by live fluorescence imaging [10,11]. Cell viability was monitored using the Cell Proliferation reagent WST-1 (Roche Applied Science) [12]. Each condition was performed in duplicate in two independent experiments. Results: The F3 and F4 hydrogels (Fig. 1A) were both capable of encapsulating rAAV as noted by effective detection of Cy3 vector labeling in the hydrogel systems relative to control conditions (encapsulation of unlabeled vectors) (Fig. 1B). An analysis of the rAAV release profiles (Fig. 1C) showed that both the F3 and F4 hydrogels effectively released rAAV over a prolonged period of time (21 days, the longest time-point evaluated) compared with hydrogels lacking rAAV vectors, with hydrogel-free rAAV vectors, and with a condition lacking both the hydrogel and rAAV. Specifically, the F4 hydrogel system was the most potent to support a controlled release of rAAV over time (Fig. 1C). An estimation of transgene (RFP) expression upon direct contact of the hydrogels with hMSCs over time revealed that rAAV-RFP released from the F3 and F4 hydrogels promoted an effective overexpression of the RFP reporter gene in the cells for at least 21 days (the longest time-point examined) versus the various control conditions tested (Fig. 2). Equally important, an analysis of the cell proliferation indices in the cells showed no deleterious effects of the F3 or F4 hydrogel systems carrying rAAV relative to the various control conditions tested (Fig. 3). Conclusions: Controlled delivery of rAAV via FID119 hydrogels (especially with the F4 hydrogel system) has the potential to safely and durably modify hMSCs. Combining rAAV gene transfer with the use of photopolymerizable (FID119) hydrogels may provide novel, promising tools to enhance cartilage repair by implanting MSCs modified to overexpress therapeutic (chondroregenerative) genes via controlled delivery of rAAV using in particular the F4 hydrogel. REFERENCES: [1] Cucchiarini & Madry, Biomed Mater Eng. 2010, 20:135 [2] Frisch et al., Curr Stem Cell Res Ther. 2015, 10:121 [3] Cottard et al., J Clin Immunol. 2004, 24:162 [4] Rey-Rico & Cucchiarini, Acta Biomater. 2016, 29:1 [5] Rey-Rico & Cucchiarini, Curr Gene Ther. 2017, 17:127 [6] Cucchiarini & Madry, Nat Rev Rheumatol. 2019, 15:18 [7] Madry et al., Adv Mater. 2020, 32:e1906508 [8] Rey-Rico et al., Biomed Res Int. 2016, 2016:1215263 [9] Meng et al., J Exp Orthop. 2019, 6:47 [10] Cucchiarini et al., Tissue Eng Part A. 2011, 17:1921 [11] Venkatesan et al., Int J Mol Sci. 2018, 19:2635 [12] Rey-Rico et al., ACS Appl Mater Interfaces. 2016, 8:20600. ACKNOWLEDGMENTS: Supported by a grant from German Osteoarthritis Foundation ( Deutsche Arthrose-Hilfe e.V. to MC).View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Effect of manganese, zinc, and copper on the biological and osteogenic properties of mesoporous bioactive glass nanoparticles.

Mesoporous bioactive glass nanoparticles (MBGNs) have demonstrated promising properties for the local delivery of therapeutically active ions with the aim to improve their osteogenic properties. Manganese (Mn), zinc (Zn), and copper (Cu) ions have already shown promising pro-osteogenic properties. Therefore, the concentration-dependent impact of MBGNs (composition in mol%: 70 SiO2 , 30 CaO) and MBGNs containing 5mol% of either Mn, Zn, or Cu (composition in mol%: 70 SiO2 , 25 CaO, 5 MnO/ZnO/CuO) on the viability and osteogenic differentiation of human marrow-derived mesenchymal stromal cells (BMSCs) was assessed in this study. Mn-doped MBGNs (5Mn-MBGNs) showed a small "therapeutic window" with a dose-dependent negative impact on cell viability but increasing pro-osteogenic features alongside increasing Mn concentrations. Due to a constant release of Zn, 5Zn-MBGNs showed good cytocompatibility and upregulated the expression of genes encoding for relevant members of the osseous extracellular matrix during the later stages of cultivation. In contrast to all other groups, BMSC viability increased with increasing concentration of Cu-doped MBGNs (5Cu-MBGNs). Furthermore, 5Cu-MBGNs induced an increase in alkaline phosphatase activity. In conclusion, doping with Mn, Zn, or Cu can enhance the biological properties of MBGNs in different ways for their potential use in bone regeneration approaches.

Administration of Human Non-Diabetic Mesenchymal Stromal Cells to a Murine Model of Diabetic Fracture Repair: A Pilot Study.

Individuals living with type 1 diabetes mellitus may experience an increased risk of long bone fracture. These fractures are often slow to heal, resulting in delayed reunion or non-union. It is reasonable to theorize that the underlying cause of these diabetes-associated osteopathies is faulty repair dynamics as a result of compromised bone marrow progenitor cell function. Here it was hypothesized that the administration of non-diabetic, human adult bone marrow-derived mesenchymal stromal cells (MSCs) would enhance diabetic fracture healing. Human MSCs were locally introduced to femur fractures in streptozotocin-induced diabetic mice, and the quality of de novo bone was assessed eight weeks later. Biodistribution analysis demonstrated that the cells remained in situ for three days following administration. Bone bridging was evident in all animals. However, a large reparative callus was retained, indicating non-union. µCT analysis elucidated comparable callus dimensions, bone mineral density, bone volume/total volume, and volume of mature bone in all groups that received cells as compared to the saline-treated controls. Four-point bending evaluation of flexural strength, flexural modulus, and total energy to re-fracture did not indicate a statistically significant change as a result of cellular administration. An ex vivo lymphocytic proliferation recall assay indicated that the xenogeneic administration of human cells did not result in an immune response by the murine recipient. Due to this dataset, the administration of non-diabetic bone marrow-derived MSCs did not support fracture healing in this pilot study.

3,5-dicaffeoyl‑epi-quinic acid from Atriplex gmelinii enhances the osteoblast differentiation of bone marrow-derived human mesenchymal stromal cells via WnT/BMP signaling and suppresses adipocyte differentiation via AMPK activation

BackgroundImpaired bone formation is one of the reasons behind osteoporosis. Alterations in the patterns of mesenchymal stromal cell differentiation towards adipocytes instead of osteoblasts contribute to osteoporosis progression. Natural anti-osteoporotic agents are effective and safe alternatives for osteoporosis treatment. PurposeIn this context, 3,5-dicaffeoyl‑epi-quinic acid (DCEQA) which is a derivative of chlorogenic acid with reported bioactivities was studied for its osteogenic differentiation enhancing potential in vitro. MethodsAnti-osteoporotic effects of DCEQA were investigated in human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) which were induced to differentiate into osteoblasts or adipocytes with or without DCEQA treatment. Changes in the osteogenic and adipogenic markers such as ALP activity and lipid accumulation, respectively, were observed along with differentiation-specific activation of mitogen activated protein kinase (MAPK) pathways. ResultsAt 10 μM concentration, DCEQA increased the proliferation of bone marrow-derived human mesenchymal stromal cells (hBM-MSCs) during osteoblast differentiation. The expression of osteogenic markers ALP, osteocalcin, Runx2, BMP2 and Wnt 10a was upregulated by DCEQA treatment. The ALP activity and extracellular mineralization were also increased. DCEQA elevated the phosphorylation levels of p38 and JNK MAPKs as well as the activation of β-catenin and Smad1/5. DCEQA suppressed the lipid accumulation and downregulated expression of adipogenic markers PPARγ, C/EBPα and SREBP1c in adipo-induced hBM-MSCs. DCEQA also decreased the phosphorylation of p38 and ERK MAPKs and stimulated the activation of AMPK in hBM-MSC adipocytes. ConclusionDCEQA was suggested to enhance osteoblast differentiation via stimulating Wnt/BMP signaling. The adipocyte differentiation inhibitory effect of DCEQA was suggested to arise from its ability to increase AMPK phosphorylation. Overall, DCEQA was shown to possess osteogenesis enhancing and adipogenesis inhibitory properties which might facilitate its use against osteoporotic conditions.

Chondrogenic potency is lost but trophic repair is maintained by highly passaged mesenchymal stem/stromal cells

Purpose: Bone marrow-derived mesenchymal stromal cells (MSCs) are defined as stem cells primarily because of their capacity for multipotent differentiation, however more recently they have been shown also to have trophic properties that may be important for promoting tissue repair through the release of regulatory molecules that can promote regeneration through the stimulation of endogenous cells, whilst also inhibiting immune/inflammatory processes. Each of these modes of action may be important when developing therapeutic strategies for the treatment of osteoarthritic joints using injectable MSCs. Since a viable MSC therapy is likely to require the use of highly expanded populations of allogenic cells, we have undertaken a detailed analysis of how the differentiation and trophic properties of MSCs change with in vitro ageing of the cells. Methods: MSCs were cultured from passage until senescence. Cells from multiple passages were tested for their potency when used either for chondrogenesis (cartilage tissue engineering assay) or trophic repair. Two methods were used as determinants of trophic repair: (i) a meniscal cartilage integration assay in which undifferentiated human MSCs on a collagen scaffold were used to induce integration of two pieces of sheep meniscal cartilage and (ii) an immunoregulation assay in which undifferentiated MSCs were used to suppress the proliferation of stimulated T-cells. Genomic and proteomic screening was used to identify potential markers of MSCS with capacity for differentiation and trophic repair. Results: MSCs from 4 different donors were found to proliferate for a minimum of 16 passages before growth arrest with cells from one donor continuing to divide even at passage 30 (Figure 1). The chondrogenic potency of these MSCs fell significantly for all four donors, when measured as either the dry weight of cartilage tissue engineering or the Glycosaminoglycans content of the engineered cartilage (Figure 2). However, there was no reduction in trophic repair whether measured as integration of meniscal cartilage or suppression of T-cell proliferation (Figure 3). Combined genomic and proteomic analysis demonstrated that earlier passage cells with both chondrogenic and trophic potency are positive for both the MMP13 gene and TIMP-1 protein, whereas later passage cells with poor chondrogenic potency but strong trophic potency are negative for the MMP13 gene but positive for the TIMP-1 protein (Figure 4). Conclusions: We have demonstrated clear differences in the regulation of chondrogenic potency and trophic properties of MSCs. The measurement of MMP13 gene and TIMP-1 protein markers will facilitate the design of future therapeutic strategies that exploit either of one these modes of action.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Treatment of vocal fold scarring with autologous bone marrow-derived human mesenchymal stromal cells\u2014first phase I/II human clinical study

BackgroundVocal fold (VF) scarring, caused by surgery or inflammation, often results in severe voice problems or aphonia. Effective lasting treatment is lacking. Previous in vitro and in vivo animal studies reported positive effects on VF scar resolution with mesenchymal stromal cell (MSC) implantation. The principal aim of this study was to examine safety aspects and secondly treatment efficacy vocal fold function in patients with VF scarring and severe voice problems.MethodsIn this open-label phase I/II study, 16 patients were treated with surgical scar resection followed by injection of autologous MSCs (0.5–2 × 106 MSCs/patient). Patients were monitored 1 year for serious adverse events (SAE) or minor complications. Therapeutic efficacy on treated VFs was evaluated by measurement of VF vibrations using high-speed laryngoscopy (HSL) and phonation pressure threshold (PTP) for elasticity and VF function. Patients self-reported voice change using the Voice Handicap Index (VHI).ResultsNo SAE or minor side effects were reported. Video ratings of VF vibrations and digitized analysis of HSL and PTP were significantly improved for 62–75% of the patients (depending on parameter). Two patients showed deteriorated VF vibrations, but improved PTP. VHI was significantly improved in 8 patients, with the remaining experiencing no significant change.ConclusionsThe results indicate that local injection of autologous MSC into scarred VFs with severe voice problems may offer a safe and feasible therapeutic option. VF vibration and elasticity were improved in approximately two thirds of treated patients.This clinical study is registered in clinicaltrials.gov (ID: NCT01981330). Retrospective registration of first patient (20130511). https//: register.clinicaltrials.gov/.

Correction to: The Expression of Immunomodulation-Related Cytokines and Genes of Adipose- and Bone Marrow-Derived Human Mesenchymal Stromal Cells from Early to Late Passages.

[This corrects the article DOI: 10.1007/s13770-018-0147-5.].

Injectable, redox-polymerized carboxymethylcellulose hydrogels promote nucleus pulposus-like extracellular matrix elaboration by human MSCs in a cell density-dependent manner.

Low back pain is a major cause for disability and is closely linked to intervertebral disc degeneration. Mechanical and biological dysfunction of the nucleus pulposus in the disc has been found to initiate intradiscal degenerative processes. Replacing or enriching the diseased nucleus pulposus with an injectable, stem cell-laden biomaterial that mimics its material properties can provide a minimally invasive strategy for biological and structural repair of the tissue. In this study, injectable, in situ-gelling carboxymethylcellulose hydrogels were developed for nucleus pulposus tissue engineering using encapsulated human marrow-derived mesenchymal stromal cells (hMSCs). With the goal of obtaining robust extracellular matrix deposition and faster construct maturation, two cell-seeding densities, 20 × 106 cells/ml and 40 × 106 cells/ml, were examined. The constructs were fabricated using a redox initiation system to yield covalently crosslinked, cell-seeded hydrogels via radical polymerization. Chondrogenic culture of the hydrogels over 35 days exhibited high cell viability along with deposition of proteoglycan and collagen-rich extracellular matrix, and mechanical and swelling properties similar to native human nucleus pulposus. Further, the matrix production and distribution in the carboxymethylcellulose hydrogels was found to be strongly influenced by hMSC-seeding density, with the lower cell-seeding density yielding a more favorable nucleus pulposus-specific matrix phenotype, while the rate of construct maturation was less dependent on the cell-seeding density. These findings are the first to demonstrate the utility of redox-polymerized carboxymethylcellulose hydrogels as hMSC carriers for potential minimally invasive treatment strategies for nucleus pulposus replacement.

The Expression of Immunomodulation-Related Cytokines and Genes of Adipose- and Bone Marrow-Derived Human Mesenchymal Stromal Cells from Early to Late Passages.

Mesenchymal stromal cells (MSCs) are multipotent stem cells that can differentiate into several cell types. In addition, many studies have shown that MSCs modulate the immune response. However, little information is currently available regarding the maintenance of immunomodulatory characteristics of MSCs through passages. Therefore, we investigated and compared cytokine and gene expression levels from adipose (AD) and bone marrow (BM)-derived MSCs relevant to immune modulation from early to late passages. MSC immunophenotype, growth characteristics, cytokine expressions, and gene expressions were analyzed. AD-MSCs and BM-MSCs had similar cell morphologies and surface marker expressions from passage 4 to passage 10. Cytokines secreted by AD-MSCs and BM-MSCs were similar from early to late passages. AD-MSCs and BM-MSCs showed similar immunomodulatory properties in terms of cytokine secretion levels. However, the gene expressions of tumor necrosis factor-stimulated gene (TSG)-6 and human leukocyte antigen (HLA)-G were decreased and gene expressions of galectin-1 and -3 were increased in both AD- and BM-MSCs with repeated passages. Our study showed that the immunophenotype and expression of immunomodulation-related cytokines of AD-MSCs and BM-MSCs immunomodulation through the passages were not significantly different, even though the gene expressions of both MSCs were different.

Mesenchymal Stromal Cells and Their Extracellular Vesicles Enhance the Anti-Inflammatory Phenotype of Regulatory Macrophages by Downregulating the Production of Interleukin (IL)-23 and IL-22.

Resolution-phase macrophage population orchestrates active dampening of the inflammation by secreting anti-inflammatory and proresolving products including interleukin (IL)-10 and lipid mediators (LMs). We investigated the effects of both human bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-derived extracellular vesicles (MSC-EVs) on mature human regulatory macrophages (Mregs). The cytokines and LMs were determined from cell culture media of Mregs cultivated with MSCs and MSC-EVs. In addition, the alterations in the expression of cell surface markers and the phagocytic ability of Mregs were investigated. Our novel findings indicate that both MSC coculture and MSC-EVs downregulated the production of IL-23 and IL-22 enhancing the anti-inflammatory phenotype of Mregs and amplifying proresolving properties. The levels of prostaglandin E2 (PGE2) were substantially upregulated in MSC coculture media, which may endorse proresolving LM class switching. In addition, our results manifest, for the first time, that MSC-EVs mediate the Mreg phenotype change via PGE2. These data suggest that both human MSC and MSC-EVs may potentiate tolerance-promoting proresolving phenotype of human Mregs.

Platelet-rich concentrate in serum free medium enhances osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells.

Previous studies have shown that platelet concentrates used in conjunction with appropriate growth media enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs). However, their potential in inducing osteogenesis of hMSCs when cultured in serum free medium has not been explored. Furthermore, the resulting osteogenic molecular signatures of the hMSCs have not been compared to standard osteogenic medium. We studied the effect of infrequent supplementation (8-day interval) of 15% non-activated platelet-rich concentrate (PRC) in serum free medium on hMSCs proliferation and differentiation throughout a course of 24 days, and compared the effect with those cultured in a standard osteogenic medium (OM). Cell proliferation was analyzed by alamar blue assay. Gene expression of osteogenic markers (Runx2, Collagen1, Alkaline Phosphatase, Bone morphogenetic protein 2, Osteopontin, Osteocalcin, Osteonectin) were analyzed using Q-PCR. Immunocytochemical staining for osteocalcin, osteopontin and transcription factor Runx2 were done at 8, 16 and 24 days. Biochemical assays for the expression of ALP and osteocalcin were also performed at these time-points. Osteogenic differentiation was further confirmed qualitatively by Alizarin Red S staining that was quantified using cetylpyridinium chloride. Results showed that PRC supplemented in serum free medium enhanced hMSC proliferation, which peaked at day 16. The temporal pattern of gene expression of hMSCs under the influence of PRC was comparable to that of the osteogenic media, but at a greater extent at specific time points. Immunocytochemical staining revealed stronger staining for Runx2 in the PRC-treated group compared to OM, while the staining for Osteocalcin and Osteopontin were comparable in both groups. ALP activity and Osteocalcin/DNA level were higher in the PRC group. Cells in the PRC group had similar level of bone mineralization as those cultured in OM, as reflected by the intensity of Alizarin red stain. Collectively, these results demonstrate a great potential of PRC alone in inducing proliferation of hMSCs without any influence from other lineage-specific growth media. PRC alone has similar capacity to enhance hMSC osteogenic differentiation as a standard OM, without changing the temporal profile of the differentiation process. Thus, PRC could be used as a substitute medium to provide sufficient pool of pre-differentiated hMSCs for potential clinical application in bone regeneration.

Independent effects of the chemical and microstructural surface properties of polymer/ceramic composites on proliferation and osteogenic differentiation of human MSCs

Within the general aim of finding affordable and sustainable regenerative solutions for damaged and diseased tissues and organs, significant efforts have been invested in developing synthetic alternatives to natural bone grafts, such as autografts. Calcium phosphate (CaP) ceramics are among widely used synthetic bone graft substitutes, but their mechanical properties and bone regenerative capacity are still outperformed by their natural counterparts. In order to improve the existing synthetic bone graft substitutes, it is imperative to understand the effects of their individual properties on a biological response, and to find a way to combine the desired properties into new, improved functional biomaterials. To this end, we studied the independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the molecular weight of the polymer and presence/absence of the ceramic phase were used as the chemical variables, a soft embossing technique was used to pattern the surfaces of all materials with either pits or pillars with identical microscale dimensions. The results indicated that, while cell morphology was affected by both the presence and availability of HA and by the surface microstructure, the effect of the latter parameter on cell proliferation was negligible. The osteogenic differentiation of hMSCs, and in particular the expression of bone morphogenetic protein 2 (BMP-2) and osteopontin (OP) were significantly enhanced when cells were cultured on the composite based on low-molecular-weight PLA, as compared to the high-molecular-weight PLA-based composite and the two pure polymers. The OP expression on the low-molecular-weight PLA-based composite was further enhanced when the surface was patterned with pits. Taken together, within this experimental set up, the individual effect of the chemistry, and in particular of the presence of CaP, was more pronounced than the individual effect of the surface microstructure, although their combined effects were, in some cases, synergistic. The approach presented here opens new routes to study the interactions of biomaterials with the biological environment in greater depths, which can serve as a starting point for developing biomaterials with improved bioactivity. Statement of SignificanceThe aim of the this study was to obtain insight into independent effects of the chemical composition and surface microstructure of a poly(lactic acid)/hydroxyapatite (PLA/HA) composite material on the morphology, proliferation and osteogenic differentiation of clinically relevant bone marrow-derived human mesenchymal stromal cells (hMSCs). While the need for synthetic alternatives for natural bone in bone regenerative strategies is rapidly increasing, the clinical performance of synthetic biomaterials needs to be further improved. To do this successfully, we believe that a better understanding of the relationship between a property of a material and a biological response is imperative. This study is a step forward in this direction, and we are therefore convinced that it will be of interest to the readers of Acta Biomaterialia.

Redefining the role of blood establishments as raw material suppliers, manufacturers, and distributors for new cell therapies: the Blood Systems experience.

Redefining the role of blood establishments as raw material suppliers, manufacturers, and distributors for new cell therapies: the Blood Systems experience.

Monolithic calcium phosphate/poly(lactic acid) composite versus calcium phosphate-coated poly(lactic acid) for support of osteogenic differentiation of human mesenchymal stromal cells.

Calcium phosphates (CaPs), extensively used synthetic bone graft substitutes, are often combined with other materials with the aim to overcome issues related to poor mechanical properties of most CaP ceramics. Thin ceramic coatings on metallic implants and polymer-ceramic composites are examples of such hybrid materials. Both the properties of the CaP used and the method of incorporation into a hybrid structure are determinant for the bioactivity of the final construct. In the present study, a monolithic composite comprising nano-sized CaP and poly(lactic acid) (PLA) and a CaP-coated PLA were comparatively investigated for their ability to support proliferation and osteogenic differentiation of bone marrow-derived human mesenchymal stromal cells (hMSCs). Both, the PLA/CaP composite, produced using physical mixing and extrusion and CaP-coated PLA, resulting from a biomimetic coating process at near-physiological conditions, supported proliferation of hMSCs with highest rates at PLA/CaP composite. Enzymatic alkaline phosphatase activity as well as the mRNA expression of bone morphogenetic protein-2, osteopontin and osteocalcin were higher on the composite and coated polymer as compared to the PLA control, while no significant differences were observed between the two methods of combining CaP and PLA. The results of this study confirmed the importance of CaP in osteogenic differentiation while the exact properties and the method of incorporation into the hybrid material played a less prominent role.

Isolation and Manufacture of Clinical-Grade Bone Marrow-Derived Human Mesenchymal Stromal Cells.

Mesenchymal stromal cells (MSCs) are multipotent cells with both regenerative and immunomodulatory capacities. These unique properties make them appealing as a biologic, with multiple phase 1-3 clinical trials currently testing their safety and efficacy. Although expanding MSCs does not require extensive manipulation, expanding MSCs for use in clinical trials does require the knowledge and safety that are delineated in current good manufacturing practices (GMPs). Here we briefly detail the characteristics of MSCs and considerations for expanding them for clinical use. We then include a step-by-step protocol for expanding MSCs for early phase clinical trials, with important notes to consider during the expansion of these MSCs.

Phenotypic, Morphological and Adhesive Differences of Human Hematopoietic Progenitor Cells Cultured on Murine versus Human Mesenchymal Stromal Cells.

Xenogenic transplantation models have been developed to study human hematopoiesis in immunocompromised murine recipients. They still have limitations and therefore it is important to delineate all players within the bone marrow that could account for species-specific differences. Here, we evaluated the proliferative capacity, morphological and physical characteristics of human CD34+ hematopoietic stem and progenitor cells (HSPCs) after co-culture on murine or human bone marrow-derived mesenchymal stromal cells (MSCs). After seven days, human CD34+CD133– HSPCs expanded to similar extents on both feeder layers while cellular subsets comprising primitive CD34+CD133+ and CD133+CD34– phenotypes are reduced fivefold on murine MSCs. The number of migrating HSPCs was also reduced on murine cells suggesting that MSC adhesion influences cellular polarization of HSPC. We used atomic force microscopy-based single-cell force spectroscopy to quantify their adhesive interactions. We found threefold higher detachment forces of human HSPCs from murine MSCs compared to human ones. This difference is related to the N-cadherin expression level on murine MSCs since its knockdown abolished their differential adhesion properties with human HSPCs. Our observations highlight phenotypic, morphological and adhesive differences of human HSPCs when cultured on murine or human MSCs, which raise some caution in data interpretation when xenogenic transplantation models are used.

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds.

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.