Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs the bovine bone matrix Nukbone (NKB) as a scaffold.
Mesenchymal stem cells (MSCs) have a differentiation potential towards osteoblastic lineage when they are stimulated with soluble factors or specific biomaterials. This work presents a novel option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) that employs bovine bone matrix Nukbone (NKB) as a scaffold. Thus, the application of MSCs in repair and tissue regeneration processes depends principally on the efficient implementation of the techniques for placing these cells in a host tissue. For this reason, the design of biomaterials and cellular scaffolds has gained importance in recent years because the topographical characteristics of the selected scaffold must ensure adhesion, proliferation and differentiation into the desired cell lineage in the microenvironment of the injured tissue. This option for the delivery of MSCs from human amniotic membrane (AM-hMSCs) employs bovine bone matrix as a cellular scaffold and is an efficient culture technique because the cells respond to the topographic characteristics of the bovine bone matrix Nukbone (NKB), i.e., spreading on the surface, macroporous covering and colonizing the depth of the biomaterial, after the cell isolation process. We present the procedure for isolating and culturing MSCs on a bovine matrix.
There pair and regeneration of tissuesis one of themain objectives of medical science because a lesion in a tissue ororgan can be incapacitating and, in some cases, lethal. In context, the health workers and researchers are constantly searching techniques, methods and tools to provide new therapeutic alternativesto promote there pair-regeneration process of damaged tissue. Therefore, biomedical sciences have focused on the study of mesenchymal stem cells (MSCs), especially adult stem cells, because this cell lineage represents an ideal model to study the tissue regeneration process, due to its potential to differentiate to ectodermal, mesodermal, and endodermal cell lineages However, to make therapy with MSCs a reality, the characterization of thetherapeutic potential of MSCs must be accompanied by the implementation of new cell culture techniques using biomaterials and cellular scaffolds that guarantee the desired behavior of the MSCs in the host tissue.
The principal challenge to overcome in the implementation of a culture with MSCs in bone regeneration is to secure cell adhesion on the selected biomaterial, which generally presents a porous structure with adistribution of pores ranging from micrometers to millimeters have been employed but frequently require sophisticated reagents, equipment, and procedures that alter the behavior of cells. These procedures are suitable for in vitro estimation, but not for the extrapolation and implementation in complex open systems, such as human or in vivo assays. Techniques that focus on ensuring cell adhesion to random topographies, such as porous bone materials, have been studied by the adhesion of the material to the bottom of culture wells and employed collagens and agarose polymers. For these methods, the cells that were seeded on the surface were retained in the pores and on the top surface. However, this aspect cannot be ensured in an in vivo system, in which the body fluids surrounding the biomaterial could drag the cells from the desired site on porous scaffolds is the use of ultrasonic stimulation, which requires sophisticated equipment but promotes the expression of osteoblastic markers in in vitro assays5. The use of continuous cultures requires bioreactors, which guarantee a homogeneous distribution of nutrients in the culture medium; however, a significant percentage of the cultured cells is lost when replacing the culture medium due to the flow rate that is used in this technique and the medium adhering to the walls of the equipment. These issues increase the cellular mass that is necessary for this type of culture and the time for obtaining a sufficient number of cells7. Thus, the methodology presented in this work represents a technique that should be extrapolated to in vivo systems because the cells are seeded in micro-mass on a top surface material, and after the appropriate incubation times, 60% of the initial cellular mass is adhered. Moreover, this technique does not require the addition of osteoblastic inductors (e.g., dexamethasone, ascorbic acid, or beta-glycerophosphate) or ultrasonic stimuli to induce osteoblastic differentiation and maintenance of the osteoblast phenotype because NKB is an osteoconductive and osteoinductive scaffolds8, the method for the culture of MSCs from the human amniotic membrane (AM-hMSCs) on macroporous bone biomaterial presented in this work representsapossibility to insert cells into damaged bone tissue and induce differentiation to the osteoblastic lineage.
This research was performed in accordance with the World Medical Association’s Declaration of Helsinki regarding the ethical conduct of research involving humans and was approved by the Research Ethic Board of Facultad de Medicina, UNAM (project number 101-2012).
1. Isolation of Human Mesenchymal Stem Cell
2. Preparation of the Porous Bone Matrix Disk (NKB)
3. Culture of Human Mesenchymal Stem Cells on the Porous Bovine Matrix
4. Cell Surface Markers Characterization by Flow Cytometry
5. Cell Adhesion Detection by Scanning Electron Microscopy
6. Cell Proliferation Assay
7. Colony Forming Unit (CFU)11
Human mesenchymal stem cell isolation
After mechanical separation of the AM from the chorion using blunt dissection (Figure 1), an adherent cell population was obtained by trypsin and collagenase II digestion. These cell populations attached in the culture dish presenta fibroblast-like cell morphology at 3 days post isolation as shown in the optical micrograph (Figure 2A) and scanning electron micrograph (Figure 2B).The placentas used in this work were obtained from healthy donor mothers withprevious informed consent.
Cell characterization by flow cytometry
The cell population that was obtained from the AM was strongly reactive to the surface mesenchymal markers CD90+ (93.5% ± 6.85) and CD73+ and CD105+ (79% ± 3.46) and showed a negative reaction to hematopoietic markers such as CD34- and CD45. Thus, the AM-hMSCs used in this work were mesenchymal, in accordancewith information previously reported by Leyva et al. (Figure 3). Additionally, this result coincides with the immunophenotype characteristics that were established by the International Society for Cellular Therapy (ISCT) for mesenchymal stem cells.
Cell adhesion on bone matrix
The scanning micrographs show the behavior of the AM-hMSCs seven days after layering on the NKB. The surface of the biomaterial was covered with spherical cells (day one), and some spreading cells apparently attached to the bovine matrix surface on the seventh day. At higher magnification, the spreading cells appeared to be in close contact via filipodial processes (FL) (Figure 4).
Cell proliferation on bone matrix
The results of the AB assay showed a small decrease in relative absorbance units (RAU) of bovine matrix condition (+bone matrix) after 1 day of incubation and then on the fifth day, the presence of the scaffold induces an increase in the cell proliferation statistically significant in comparison with the culture performed in the absence of bovine matrix (-bone matrix) (Figure 5).
Colony Forming Unit
The CFU is a traditionally assay of stem cells. The AM-hMSCs isolated in this work preserved its capability to form discrete colonies at 14 days in culture.
Figure 1: Isolation of amniotic membrane. (A) The umbilical cord (UC) is held with one hand to identify the region in which the amniotic membrane is obtained. (B) The amniotic membrane (AM) resembles a translucent film without blood innervations.
Figure 2: Morphological characteristics of stem cells from the human amniotic membrane. AM-hMSC cultures at 3 days post-isolation were observed using an optical microscope and shown the fibroblast-like morphology.
Figure 3: Flow cytometry characterization. The cells from human amniotic membrane were mostly mesenchymal cells because they were positive for mesenchymal markers (CD73, CD90, CD105), as shown by the displacement of histogram to the right, and negative for hematopoietic markers (CD34 and CD45), as confirmed by the displacement of the histogram to the left. The antigens are shown in red histograms, while the control isotypesfor the PE and FITC fluorochromes are observed in black.
Figure 4: Cell adhesion on bovine bone matrix. A series of SEM images showing attachment of the cells to pores of the biomaterial. (A) Panoramic vision of a NKB pore with several cells adhered to the biomaterial in the spherical state (CS) and flattened (CF) on the surface of the biomaterial, is also possible to appreciate the bone lacuna indicates as (Lac) at three day of culture on the bovine matrix. (B) Amplification of the cell in the process of adapting at the surface of the material through filipodial projections. (C) Interaction between two cells adhered and flattened on the bovine matrix (C1, cell 1; and C2, cell 2). Please click here to view a larger version of this figure.
Figure 5: Proliferation of cells on bovine bone matrix. Cell proliferation was evaluated by AB reduction and expressed in relative absorbance units along 7 days of cultivation. Results are shown the influence of bone matrix (+bone matrix) in AM-hMSCs proliferation which was statically different in comparison with the negative condition (-bone matrix). (*p <0.05)
Figure 6: CFU assay of MSCs initially plated at varying densities and incubated for 14 days (mean*/- SD, n = 10).
Cells adhered | Cell adhesion (%) | |
Cells on the bottom | 187667 ± 1208 | 62.47 |
Cells on the scaffold | 312333 ±1208 | 37.53 |
Table 1: Efficiency of adhesion on bovine matrix. The cells adhered to the bottom of the plate dish and the cells adhering on the scaffold were counted by hemocytometer after being recovered by trypsinization.The data presented in the table correspond to two experiment independent for triplicate ± standard deviation
The stem cells that were obtained from human amniotic membrane (Figure 1) showed a fibroblast-like morphology, similar to mesenchymal cells (Figure 2) and were principally MSCs. In addition, flow cytometry assays revealed that these cells were positive for mesenchymal cell markers (CD73, CD90, and CD105) and negative for hematopoietic lineage markers (CD34, CD45) (Figure 3). Also, the AM-hMSCs isolated in this work present the capability of forming CFUs (Figure 6). Similarly, the mesenchymal stem cells isolated in this form retained the ability to attach to osteoinductor biomaterial (Figure 4), and proliferate on the scaffold (Figure 5).
For these reasons, the culture method of AM-hMSCs on macroporous bone biomaterial that was used in this work represents an opportunity to insert cells within injured bone tissue and induce its differentiation to the osteoblastic lineage. This culture system does not require the addition of osteoblastic inductors to maintain the osteoblast phenotype and osteoblastic differentiation process because NKB is an osteoconductive andosteoinductive material8. The use of waste tissues, such as human placenta, represents a simple and ethical alternative to access a population of stem cells with the potential to differentiate into MSCs, in comparison with other sources that frequently involve invasive methods or low cell yield.
The proposed culture method could be extrapolated to in vivo systems because the cells are seeded in micro-mass on the top surface material, and the cells colonize and adhere to the entire biomaterial surface after the appropriate incubation times. Furthermore, sophisticated reagents, equipment and procedures are not required, unlike existing methods when implementing this technique are the micro-mass cultureas well as the slow and steady deposition of the cellular aliquot on the material to ensure that the cells preferentially interact with the biomaterial, and not with the bottom of the plate. Another critical point is the use of pre-wetted material before culturing the cells, as this ensures that the cells will have the necessary nutrients from the culture medium when they are integrated into the material regardless if they are on the surface or within the pores. Finally, the use of an osteoinductive biomaterial is critical to this process because it avoids the addition of external factors to induce and maintain osteoblastic differentiation. The limitation of the technique is that it is based on the osteoinductive potential of the bovine matrix; however, the proposed culture system can be extrapolated to any porous material with a pore distribution of 20 to 200 µm. Therefore, the procedure presented in this work is an alternative method for culturing mesenchymal stem cells in various materials that are employed for tissue engineering, especially for bone regeneration.
The authors have nothing to disclose.
We acknowledge the scholarship and financial support provided by the Consejo Nacional de Ciencia y Tecnología (CONACyT No.49887), Facultad de Medicina-UNAM DGAPA IN201510 and DGAPA IN216213 ; Salvador Correa of Instituto Nacional de Perinatología for help with the biological material; and Biocriss, S. A. de C. V for donating Nukbone. Also thank to Ing. Héctor Martínez of IIM, UNAM and M en C Fabiola Gonzalez of CINVESTAV for technical assistance.
sterile phosphate buffered saline | GIBCO,LIFE TECHNOLOGIES | 10010023 | cell culture |
penicillin-streptomycin | GIBCO,LIFE TECHNOLOGIES | 10378016 | cell culture |
FBS | GIBCO,LIFE TECHNOLOGIES | 26140111 | cell culture |
Dulbecco's Modified Eagle's Medium (DMEM) with high glucose | GIBCO,LIFE TECHNOLOGIES | 11965118 | cell culture |
collagenase type II | GIBCO,LIFE TECHNOLOGIES | 17101015 | cell culture |
3mM calcium chloride | SIGMA_ALDRICH | C5670 | cell culture |
trypsin/0.5mM EDTA solution | GIBCO,LIFE TECHNOLOGIES | R001100 | cell culture |
Trypan Blue solution | GIBCO,LIFE TECHNOLOGIES | 15250061 | cell culture |
phycoerythrin (PerCP)-conjugated CD73 | BD Pharmigen | 562245 | cytomery |
FITC- CD90, | BD Pharmigen | 562245 | cytomery |
PE-CD34 | BD Pharmigen | 562245 | cytomery |
FITC-CD45 | BD Pharmigen | 562245 | cytomery |
PE-CD105 | BD Pharmigen | 562245 | cytomery |
BD FACSCalibur Flow Cytometer | BD Pharmigen | BD FACSCalibur | cytomery |
alamarBlue (AB) | LIFE TECHNOLOGIES | DAL1025 | proliferation cell assay |
SUNRISE-reader | TECAN, Germany | Sunrise | proliferation cell assay |