Figure 4 - available from: Scientific Reports
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Representative images of in vitro osteogenic (Left: Alizarin Red S staining, magnification 4×; Right: LIVE/DEAD fluorescent staining, magnification 10×), adipogenic (Left: Oil Red O, magnification 40×; Right: LIVE/DEAD fluorescent staining, magnification 10×) and chondrogenic (Left: Alcian Blue/Nuclear Fast Red staining, magnification 80×; Right: Collagen Type II magnification 80×) differentiation of MSCs from (a) whole, (b) concentrated and (c) clotted BMAs. Black arrows: chodrocytes; Red arrows: extracellular matrix.
Source publication
Vertebral body bone marrow aspirate (V-BMA), easily accessible simultaneously with the preparation of the site for pedicle screw insertion during spinal procedures, is becoming an increasingly used cell therapy approach in spinal surgery. However, the main drawbacks for V-BMA use are the lack of a standardized procedure and of a structural texture...
Contexts in source publication
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... vitro osteogenic, adipogenic and chondrogenic differentiation. In all culture conditions, LIVE/DEAD staining confirmed the viability of MSCs exposed to osteogenic and adipogenic medium (Fig. 4). Osteogenic induced-MSCs derived from whole, concentrated and clotted V-BMAs showed a change of cells shape and presence of mineralized matrix (red staining). However, in MSCs from whole and clotted V-BMAs a greater amount of calcium deposits were seen and this aspect was more marked in clotted V-BMA (Fig. 4). A decrease in cell ...
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... to osteogenic and adipogenic medium (Fig. 4). Osteogenic induced-MSCs derived from whole, concentrated and clotted V-BMAs showed a change of cells shape and presence of mineralized matrix (red staining). However, in MSCs from whole and clotted V-BMAs a greater amount of calcium deposits were seen and this aspect was more marked in clotted V-BMA (Fig. 4). A decrease in cell density and a change of cells shape, that become more elongated and flat, were observed in all culture conditions when MSCs were exposed to adipogenic medium. The reduction in cell density was more evident in particular for MSCs from whole and clotted V-BMAs (Fig. 4). Concerning the chondrogenic differentiation of ...
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... were seen and this aspect was more marked in clotted V-BMA (Fig. 4). A decrease in cell density and a change of cells shape, that become more elongated and flat, were observed in all culture conditions when MSCs were exposed to adipogenic medium. The reduction in cell density was more evident in particular for MSCs from whole and clotted V-BMAs (Fig. 4). Concerning the chondrogenic differentiation of MSCs, in all culture conditions the presence of chondrocytes inside the lacunae separated by extracellular matrix was seen (Fig. 4). Although in all culture conditions MSCs differentiated towards the chondrogenic phenotype, MSCs from clotted V-BMA highlighted a greater amount of ...
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... all culture conditions when MSCs were exposed to adipogenic medium. The reduction in cell density was more evident in particular for MSCs from whole and clotted V-BMAs (Fig. 4). Concerning the chondrogenic differentiation of MSCs, in all culture conditions the presence of chondrocytes inside the lacunae separated by extracellular matrix was seen (Fig. 4). Although in all culture conditions MSCs differentiated towards the chondrogenic phenotype, MSCs from clotted V-BMA highlighted a greater amount of chondrocytes that were also organized in isogenous group (Fig. 4). Immunostaining showed the presence of Collagen Type II in all MSCs micromasses with more prominent staining in MSCs ...
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... of MSCs, in all culture conditions the presence of chondrocytes inside the lacunae separated by extracellular matrix was seen (Fig. 4). Although in all culture conditions MSCs differentiated towards the chondrogenic phenotype, MSCs from clotted V-BMA highlighted a greater amount of chondrocytes that were also organized in isogenous group (Fig. 4). Immunostaining showed the presence of Collagen Type II in all MSCs micromasses with more prominent staining in MSCs derived from whole and clotted V-BMAs, while the staining was less evident in MSCs derived from concentrated V-BMA (Fig. ...
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... from clotted V-BMA highlighted a greater amount of chondrocytes that were also organized in isogenous group (Fig. 4). Immunostaining showed the presence of Collagen Type II in all MSCs micromasses with more prominent staining in MSCs derived from whole and clotted V-BMAs, while the staining was less evident in MSCs derived from concentrated V-BMA (Fig. ...
Citations
... Various properties of BMSCs, such as proliferation, differentiation, clonogenic potential, and surface antigen expression, have been proven to be affected by overweight or obesity in animals and humans 12,13,27,28 . Notably, these properties are important criteria for the clinical application of BMSCs [29][30][31] . However, current research results on these aspects are contradictory. ...
Bone marrow–derived mesenchymal stromal cells (BMSCs) are attractive candidates in tissue engineering and regenerative medicine. Growing evidence has suggested that a high body mass index (BMI) can affect the properties of BMSCs, resulting in a reduced quality of the cells. However, the results are not consistent. Therefore, this study aimed to investigate the influences of high BMI on human BMSCs (hBMSCs). To avoid gender bias, BMSCs from females and males were studied independently. Finally, hBMSCs from 89 females and 152 males were separately divided into the normal BMI group (18.5 kg/m² ≤ BMI < 25 kg/m²) and the high BMI group (BMI > 25 kg/m²). The cells were analyzed for the colony-forming potential; proliferation capacity; in vitro adipogenic, osteogenic, and chondrogenic differentiation potentials; and the expression of 32 common surface antigens. The results showed that high BMI did not change the number of colonies at passage 1 in females and males. In contrast, significantly reduced colony numbers at passage 4 (P4) were found in both female and male donors with high BMI. The doubling time of hBMSCs was comparable between the normal and the high BMI groups of females and males. Furthermore, the results of trilineage differentiation did not differ between the different BMI groups of males. In females, the high and the normal BMI groups also showed similar adipogenic and chondrogenic differentiation, while osteogenic differentiation was significantly enhanced in the high-BMI group. Regarding the expression of surface antigens, the expressions of CD200 and SSEA4 on hBMSCs were reduced in the high-BMI group of females and males, respectively. In conclusion, high BMI suppressed the clonogenicity of female and male hBMSCs at P4, improved the in vitro osteogenesis of female hBMSCs, and decreased the expressions of CD200 on hBMSCs in females and SSEA4 in males.
... However, its use is restricted by the absence of an effective processing method and a structural texture and by the possibility of dispersion away from the implant site. In 2018, our group described for the first time the use of a new formulation of vBMA, the vBMA clot (17); MSCs from human clotted vBMA exhibited higher kinetics of growth in comparison to MSCs from human unclotted vBMA as well as higher growth factor expression, higher osteogenic and chondrogenic differentiation ability, and lower expression of TALE and HOX genes, classes of genes that harmfully control osteoblast proliferation, differentiation, and maturation (18). This result was replicated among elderly and super-elderly patients, in whom it was revealed that donor age does not disturb tissuespecific vBMA clot regenerative properties nor the expression of the KLOTHO gene (aging suppressor gene) or senescence-associated genes (18). ...
... This result was replicated among elderly and super-elderly patients, in whom it was revealed that donor age does not disturb tissuespecific vBMA clot regenerative properties nor the expression of the KLOTHO gene (aging suppressor gene) or senescence-associated genes (18). Collectively, these studies (15)(16)(17)(18) suggested that the vBMA clot may play an important role as an osteogenic and osteoinductive 3D bioscaffold, being an abundant source of mesenchymal and hematopoietic stem cells working in a synergic manner to promote bone regeneration. The clotted vBMA use not only removes the need to concentrate and/or purify vBMA but also gives a smart spinal fusion cell therapy approach able to offer high "stability" to the graft site. ...
... This has previously been attributed to the lack of an effective BMA processing method and a structural texture and to the possibility of dispersion away from the implant site. Recently, for the first time, the potential use of a powerful formulation of BMA, the BMA clot, was described by our team (15,17,18). It comprises a clot naturally formed from vertebral bone marrow, which has all the vBMA elements preserved in a matrix molded by the clot. ...
Background
Bone marrow aspirate (BMA), when combined with graft substitutes, has long been introduced as a promising alternative to iliac crest bone graft in spinal fusion. However, the use of BMA is limited by the absence of a standardized procedure, a structural texture, and the potential for diffusion away from the implant site. Recently, the potential use of a new formulation of BMA, named BMA clot, has been preclinically described. In this report, we present the results of a prospective pilot clinical study aimed at evaluating the safety and efficacy of autologous vertebral BMA (vBMA) clot as a three-dimensional and multifunctional bioscaffold in instrumented posterior lumbar fusion.
Methods
Ten consecutive patients with an indication of multilevel (≤5) posterior spinal fusion due to lumbar spine degenerative diseases were included in the study and treated with vBMA. Clinical outcomes were assessed using the Visual Analog Scale (VAS), Oswestry Disability Index (ODI), and EuroQoL-5L (EQ-5L) preoperatively and at 3 months and 12 months after spinal fusion. Bone fusion quality was evaluated at the 12-month follow-up using the Brantigan classification on radiography (XR) imaging. Bone density was measured on computed tomography (CT) scans at 6 and 12 months of follow-up visits at the intervertebral arches and intervertebral joint areas and expressed in Hounsfield unit (HU).
Results
The results indicate a successful posterolateral fusion rate of approximately 100% (considering levels with C, D, and E grades according to the Brantigan classification) at the 12-month follow-up, along with an increase in bone density from 6 to 12 months of follow-up. An improvement in the quality of life and health status following surgery, as assessed by clinical scores (ODI, VAS, and EQ-5L), was also observed as early as 3 months postsurgery. No adverse events related to the vBMA clot were reported.
Conclusion
This prospective pilot study demonstrates the effectiveness and safety profile of vBMA clot as an advanced bioscaffold capable of achieving posterior lumbar fusion in the treatment of degenerative spine diseases. This lays the groundwork for a larger randomized clinical study.
... These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials. attributed to its three-dimensional (3D) matrix, which facilitates the delivery of mesenchymal stem cells (MSCs) and alpha granules, platelet-specific proteins, cytokines/chemokines, growth factors, coagulation factors, and adhesion molecules [12,13]. To validate the clinical use of vBMA clots for spinal bone regeneration, a pilot clinical study is currently underway at our institute (CE-AVEC 587/2020/Sper/IORS). ...
Recently, our group described the application of vertebral bone marrow (vBMA) clot as a cell therapy strategy for spinal fusion. Its beneficial effects were confirmed in aging-associated processes, but the influence of gender is unknown. In this study, we compared the biological properties of vBMA clots and derived vertebral mesenchymal stem cells (MSCs) from female and male patients undergoing spinal fusion procedures and treated with vBMA clot. We analyzed the expression of growth factors (GFs) in vBMA clots and MSCs as well as morphology, viability, doubling time, markers expression, clonogenicity, differentiation ability, senescence factors, Klotho expression, and HOX and TALE gene profiles from female and male donors. Our findings indicate that vBMA clots and derived MSCs from males had higher expression of GFs and greater osteogenic and chondrogenic potential compared to female patients. Additionally, vBMA-clot-derived MSCs from female and male donors exhibited distinct levels of HOX and TALE gene expression. Specifically, HOXA1, HOXB8, HOXD9, HOXA11, and PBX1 genes were upregulated in MSCs derived from clotted vBMA from male donors. These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials.
... Clonogenicity is a common method to assess the self-renewal of mesenchymal stem cells (MSCs) [54]. Nowadays, colony formation efficiency is often employed to evaluate the quality of MSC preparations for preclinical studies and clinical trials [55,56]. In animal research, high-fat diet (HFD)-induced obesity best mimics the physiological functions of the obese human body [33,57]. ...
Bone marrow-derived mesenchymal stem cells (BMSCs) are promising candidates for cell-based therapies. Growing evidence has indicated that overweight/obesity can change the bone marrow microenvironment, which affects some properties of BMSCs. As the overweight/obese population rapidly increases, they will inevitably become a potential source of BMSCs for clinical application, especially when receiving autologous BMSC transplantation. Given this situation, the quality control of these cells has become particularly important. Therefore, it is urgent to characterize BMSCs isolated from overweight/obese bone marrow environments. In this review, we summarize the evidence of the effects of overweight/obesity on the biological properties of BMSCs derived from humans and animals, including proliferation, clonogenicity, surface antigen expression, senescence, apoptosis, and trilineage differentiation, as well as the underlying mechanisms. Overall, the conclusions of existing studies are not consistent. Most studies demonstrate that overweight/obesity can influence one or more characteristics of BMSCs, while the involved mechanisms are still unclear. Moreover, insufficient evidence proves that weight loss or other interventions can rescue these qualities to baseline status. Thus, further research should address these issues and prioritize developing methods to improve functions of overweight- or obesity-derived BMSCs.
... Therefore, several methods, such as graft materials, cells and growth factors have been intensively investigated to enhance SF. Lately, our research group, for the first time, evaluated the possibility of using a new and advanced formulation of vertebral bone marrow (vBM), the vBM clot [3][4][5]. It consists of a clot naturally formed from bone marrow, which has all the vBM components retained in a matrix molded by the clot. ...
... It consists of a clot naturally formed from bone marrow, which has all the vBM components retained in a matrix molded by the clot. In detail, mesenchymal stem cells (MSCs) from human clotted vBM showed significantly higher growth kinetics in comparison to MSCs from un-clotted vBM as well as greater growth factor expression (e.g., transforming growth factor-β, TGF-β; vascular endothelial growth factor-A, VEGF-A; fibroblast growth factor 2, FGF2), higher osteogenic and chondrogenic differentiation ability, and lower expression of Meis3 and Pbx1 genes, TALE and HOX class genes that negatively regulate the induction, proliferation, differentiation, and maturation of osteoblasts [4]. These results suggest that the cellular source inside the clotted vBM have the best biological properties. ...
... In addition to this regenerative capacity proved by the vBM clot, we believe that this autologous bioscaffold may be able to target all the main key challenges for SF surgery. First, the vBM clot acts as an osteogenic and osteoinductive three-dimensional (3D) bioscaffold containing pluripotent mesenchymal and hematopoietic stem cell that work synergistically to foster bone formation and regeneration [5]; second, platelets degranulation in the vBM clot allow the release of many biomolecules (αgranules, platelet-specific proteins, cytokines/chemokines, growth factors, coagulation factors, adhesion molecules) that might promote an early vascularization, vital for bone homeostasis, healing, regeneration and hardware osseointegration [3,5]; third, mesenchymal progenitors in vBM clot modulates inflammation through a paracrine immunomodulatory effect allowing an optimal transient stage of acute inflammation, key element for a successful bone healing [3]; fourth, an additional powerful element of vBM clot could be represented by the critical role of the coagulation cascade and of the bone marrow mesenchymal stem cells (BMSCs) in the early innate immune system activation and in its involvement in stressing and eliminating bacteria [3][4][5]. We assume that the ability of vBM clot to provide a local combined delivery system of stem cells, signaling biomolecules and anti-inflammatory and antibacterial factors enclosed by a matrix molded by the clot represent an advanced and simple strategy to meet the main clinical needs of SF. ...
Exploring innovative techniques and treatments to improve spinal fusion procedures is a global challenge. Here, we provide a scientific opinion on the ability of a vertebral bone marrow (vBM) clot to provide a local combined delivery system not only of stem cells, signaling biomolecules and anti-inflammatory factors but also of molecules and proteins endowed with antimicrobial properties. This opinion is based on the evaluation of the intrinsic basic properties of the vBM, that contains mesenchymal stem cells (MSCs), and on the coagulation process that led to the conversion of fibrinogen into fibrin fibers that enmesh cells, plasma but above all platelets, to form the clot. We emphasize that vBM clot, being a powerful source of MSCs and platelets, would allow the release of antimicrobial proteins and molecules, mainly cathelicidin LL- 37, hepcidin, kinocidins and cationic host defense peptides, that are per se gifted with direct and/or indirect antimicrobial effects. We additionally highlight that further studies are needed to deepen this knowledge and to propose vBM clot as multifunctional bioscaffold able to target all the main key challenges for spinal fusion surgery.
... Although only 0.01-0.001% of MSCs is found among the totality of mononuclear cells in BMA, the concurrent presence of nonadherent osteogenic cells and the establishment of cell-cell interactions suggest that the use of whole BMA, instead of BMC or expanded and purified MSCs, is preferable for bone cell therapy (Hernigou et al., 2005). Recently, our research group also demonstrated that MSCs derived from human clotted BMA have higher growth kinetics in comparison to MSCs derived from human un-clotted BMA (whole and concentrate) as well as higher growth factors expression (transforming growth factor beta, TGF-β; vascular-endothelial growth factor, VEGF-A; fibroblast growth factor 2, FGF2) and higher ability to differentiate toward the osteogenic and chondrogenic phenotype (Salamanna et al., 2020). These data were also confirmed by an in vivo study by Lim and colleagues that underlined that the therapeutic potential of autologous bone graft and BMA clot in the repair of ulnar defects are similar, with the advantage of BMA clot being associated with a lower risk of complications (Lim et al., 2019). ...
... Considering that MSCs from clotted vertebral BMA showed better biological properties in comparison to the whole and concentrated BM, it represents a point-of-care orthobiologic product that uniquely and simultaneously delivers growth factors and MSCs (Salamanna et al., 2020). The goal of this study was to evaluate the influence of donor age on human vertebral clotted BMA, one of the skeletal sites with the highest bone turnover, characterizing and comparing the biological properties of MSCs from younger (<45 years) and older female donors (>65 years). ...
... The formation of a BMA clot comprises the degranulation of platelets that deliver various osteotropic cytokines and GFs, such as PDGF-AB, FGF, and BMPs, as well as angiogenic factors derived by the fibrinolytic activity occurring within the clot (Muschler et al., 2003;Palta et al., 2014). Recent studies demonstrated that MSCs from the clotted BMA showed enhanced biological properties in comparison to concentrate and whole BM and that the clotted BMA is an effective and efficient alternative option to autograft for in vivo long bone healing (Lim et al., 2019;Salamanna et al., 2020). Thanks to its properties, the BMA clot behaves like an early hematoma, able not only to deliver new cells to the injury site but also to trigger an immunomodulatory cascade capable of attracting peripheral MSCs to the injury site. ...
Recently, the use of a new formulation of bone marrow aspirate (BMA), the BMA clot, has been described. This product entails a naturally formed clot from the harvested bone marrow, which retains all the BMA components preserved in a matrix biologically molded by the clot. Even though its beneficial effects were demonstrated by some studies, the impact of aging and aging-associated processes on biological properties and the effect of BMA cell-based therapy are currently unknown. The purpose of our study was to compare selected parameters and properties of clotted BMA and BMA-derived mesenchymal stem cells (MSCs) from younger (<45 years) and older (>65 years) female donors. Clotted BMA growth factors (GFs) expression, MSCs morphology and viability, doubling time, surface marker expression, clonogenic potential, three-lineage differentiation, senescence-associated factors, and Klotho synthesis from younger and older donors were analyzed. Results indicated that donor age does not affect tissue-specific BMA clot regenerative properties such as GFs expression and MSCs morphology, viability, doubling time, surface antigens expression, colony-forming units, osteogenic and adipogenic differentiation, and Klotho and senescence-associated gene expression. Only few differences, i.e., increased platelet-derived growth factor-AB (PDGF-AB) synthesis and MSCs Aggrecan (ACAN) expression, were detected in younger donors in comparison with older ones. However, these differences do not interfere with all the other BMA clot biological properties. These results demonstrated that BMA clot can be applied easily, without any sample processing and avoiding potential contamination risks as well as losing cell viability, proliferation, and differentiation ability, for autologous transplantation in aged patients. The vertebral BMA clot showed two successful hits since it works as a biological scaffold and as a powerful source of mesenchymal stem cells, thus representing a novel and advanced therapeutic alternative for the treatment of orthopedic injuries.
... The CFU assay measures the efficiency by which MSCs form colony units when plated at clonogenic levels in monolayer culture on tissue culture plastic. Today, colony-forming efficiency is frequently employed for quality assessment of MSC preparations used in preclinical research and clinical trials [2,3]. ...
Colony-forming efficiency is a time-honored metric of the proliferation potential of mesenchymal stem cells (MSCs). This commentary raises a concern about the practice of using colony-forming efficiency as a proxy for cell survival. A recently published study from my laboratory investigated this issue. A marker of cellular aging, CD264, was employed to separate human bone marrow MSCs into populations of CD264− cells and culture-matched, aging CD264+ cells with high and low colony-forming efficiency, respectively. In vitro cell survival was evaluated with a single-cell assay; in vivo survival by bioluminescence imaging of MSCs attached to scaffolds that were implanted ectopically in immunodeficient mice. In our study, in vitro and in vivo survival of the MSC populations was independent of colony-forming efficiency. This finding indicates that caution should be exercised before using colony-forming efficiency as an indirect metric of cell survival. Direct measurement of survival may be required. Awareness of this issue should foster a robust experimental design and, thereby, facilitate the translation of MSC research into clinical practice.
