Streamlining the generation of an osteogenic graft by 3D culture of unprocessed bone marrow on ceramic scaffolds

Mesenchymal stromal cells are present in very low numbers in the bone marrow, necessitating their selective expansion on tissue culture plastic prior to their use in tissue-engineering applications. MSC expansion is laborious, time consuming, unphysiological and not economical, thus calling for automated bioreactor-based strategies. We and others have shown that osteogenic grafts can be cultured in bioreactors by seeding either 2D-expanded cells or by direct seeding of the mononuclear fraction of bone marrow. To further streamline this protocol, we assessed in this study the possibility of seeding the cells onto porous calcium phosphate ceramics directly from unprocessed bone marrow. Using predetermined volumes of bone marrow from multiple human donors with different nucleated cell counts, we were able to grow a confluent cell sheath on the scaffold surface in 3 weeks. Cells of stromal, endothelial and haematopoietic origin were detected, in contrast to grafts grown from 2D expanded cells, where only stromal cells could be seen. Upon implantation in nude mice, similar quantities of bone tissue were generated as compared to that obtained by using the conventional number of culture expanded cells from the same donor. We conclude that human osteogenic grafts can be efficiently prepared by direct seeding of cells from unprocessed bone marrow

The effect of bone marrow aspiration strategy on the yield and quality of human mesenchymal stem cells

Introduction Large inter-donor differences exist in human mesenchymal stem cell (hMSC) yield and the response of these cells to osteogenic stimuli. The source of these differences may be clinical differences in stem cell characteristics between individuals or the aspiration procedure itself

A link between the accumulation of DNA damage and loss of multi-potency of human mesenchymal stromal cells

Human mesenchymal stromal cells (hMSCs) represent an attractive cell source for clinic applications. Besides being multi-potent, recent clinical trials suggest that they secrete both trophic and immunomodulatory factors, allowing allogenic MSCs to be used in a wider variety of clinical situations. The yield of prospective isolation is however very low, making expansion a required step toward clinical applications. Unfortunately, this leads to a significant decrease in their stemness. To identify the mechanism behind loss of multi-potency, hMSCs were expanded until replicative senescence and the concomitant molecular changes were characterized at regular intervals. We observed that, with time of culture, loss of multi-potency was associated with both the accumulation of DNA damage and the respective activation of the DNA damage response pathway, suggesting a correlation between both phenomena. Indeed, exposing hMSCs to DNA damage agents led to a significant decrease in the differentiation potential. We also showed that hMSCs are susceptible to accumulate DNA damage upon in vitro expansion, and that although hMSCs maintained an effective nucleotide excision repair activity, there was a progressive accumulation of DNA damage. We propose a model in which DNA damage accumulation contributes to the loss of differentiation potential of hMSCs, which might not only compromise their potential for clinical applications but also contribute to the characteristics of tissue agein

Engineering New Bone via a Minimally Invasive Route Using Human Bone Marrow-Derived Stromal Cell Aggregates, Microceramic Particles, and Human Platelet-Rich Plasma Gel

There is a rise in the popularity of arthroscopic procedures in orthopedics. However, the majority of cell based bone tissue engineered constructs rely on solid pre-formed scaffolding materials, which require large incisions and extensive dissections for placement at the defect site. Thus, they are not suitable for minimally invasive techniques. The aim of this study was to develop a clinically relevant, easily moldable, bone tissue engineered construct (TEC), amenable to minimally invasive techniques, using human mesenchymal stromal cells (hMSC) and calcium phosphate micro particles in combination with an in-situ forming platelet rich plasma (PRP) gel obtained from human platelets. Most conventional TECs rely on seeding and culturing single cell suspensions of hMSCs on scaffolds. However, for generating TECs amenable to the minimally invasive approach, it was essential to aggregate the hMSCs in vitro prior to seeding them on the scaffolds as unaggregated MSCs did not generate any bone. 24 hours of in vitro aggregation was determined to be optimal for maintaining cell viability in vitro and bone formation in vivo. Moreover, no statistically significant difference was observed in the amount of bone formed when the TECs were implanted via an open approach or a minimally invasive route. TECs generated using MSCs from three different human donors generated new bone through the minimally invasive route in a reproducible manner, suggesting that these TECs could be a viable alternative to pre-formed scaffolds employed through an open surgery for treating bone defects

Additional file 4: Figure S3. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Surface marker expression (in percentage) of the acoustic stimulated cells represented as a bar plot. Each bar represents the average expression obtained from three independent donors. Represented are only the surface markers that were expressed in the obtained populations. Negative markers are not shown. No statistically significant differences were found between the two conditions. (PDF 184 kb

Additional file 3: Figure S2. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Biological characterization of isolated hMSCs from acoustically stimulated BM at 300 Hz for 5 min at different volumes, 11.5, 10, 8, 6 and 5 ml. The results are presented as the fold change over the non-stimulated bone marrow (baseline). (A) Graphic representation of the bone marrow volumes, donor dependent. (B) Proliferation of hMSCs calculated as PD/day from P1 to P2, donor and volume dependent. (C) CFU potential of hMSCs, donor and volume dependent. (D) ECM production, quantification of nodule size area in mm2, donor and volume dependent. (E) Osteogenic potential calculated as percentage of ALP positive colonies within the CFUs, donor and volume dependent. (F) Adipogenic potential, quantification of Oil red O staining relative to 100% Oil red O staining solution, donor and volume dependent. Values are represented as mean ± standard deviation of at least three independent experiments (n ≥ 3). Statistically significant differences were found with ***p < 0.001, **p < 0.01 and *p < 0.05. (PDF 694 kb

Additional file 5: Figure S4. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Alizarin red staining of calcium nodules after osteogenic induction of hMSC isolated under varying culture condition from different donors. No differences were observed between the culture conditions, though differences between the donors were identified. Donor 2 and 11 showed less calcium nodules formation than the rest of the donors. All the controls stained negative for calcium nodules formation. Values are represented as mean ± standard deviation of at least three independent experiments (n = 3). (PDF 2096 kb

Additional file 2: Figure S1. of Means of enhancing bone fracture healing: optimal cell source, isolation methods and acoustic stimulation

Macroscopic appearance of bone marrow aspirated from different locations: ilium, proximal femur, distal femur and proximal tibia. (PDF 311 kb

Means of enhancing bone fracture healing: Optimal cell source, isolation methods and acoustic stimulation

Background: The human body has an extensive capacity to regenerate bone tissue after trauma. However large defects such as long bone fractures of the lower limbs cannot be restored without intervention and often lead to nonunion. Therefore, the aim of the present study was to assess the pool and biological functions of human mesenchymal stromal cells (hMSCs) isolated from different bone marrow locations of the lower limbs and to identify novel strategies to prime the cells prior to their use in bone fracture healing. Following, bone marrow from the ilium, proximal femur, distal femur and proximal tibia was aspirated and the hMSCs isolated. Bone marrow type, volume, number of mononuclear cells/hMSCs and their self-renewal, multilineage potential, extracellular matrix (ECM) production and surface marker profiling were analyzed. Additionally, the cells were primed to accelerate bone fracture healing either by using acoustic stimulation or varying the initial hMSCs isolation conditions. Results: We found that the more proximal the bone marrow aspiration location, the larger the bone marrow volume was, the higher the content in mononuclear cells/hMSCs and the higher the self-renewal and osteogenic differentiation potential of the isolated hMSCs were. Acoustic stimulation of bone marrow, as well as the isolation of hMSCs in the absence of fetal bovine serum, increased the osteogenic and ECM production potential of the cells, respectively. Conclusion: We showed that bone marrow properties change with the aspiration location, potentially explaining the differences in bone fracture healing between the tibia and the femur. Furthermore, we showed two new priming methods capable of enhancing bone fracture healing