An in-vitro adhesion pattern study of Hematopoietic Stem Cells on porous titanium and bioceramic scaffolds.
Abstract: Hematopoietic Stem Cells (HSCs) are self-renewing multipotent cells able to produce all blood cell lineages. More relevant to Hematopoietic Stem Progenitor Cells (HSPC) expansion, studies have suggested that a 3D culture microenvironment is conducive for HSPC proliferation and differentiation. The present study is an attempt to establish the conditions for ex vivo expansion and long term culture of HSCs on porous bioceramic (Tricalcium phosphate, TCP) and titanium (Ti) scaffolds in order to sustain durable, long-term hematopoiesis. The Ti scaffolds (40 vol.% porosity) have been prepared through powder metallurgy route with pore size distribution in the range of 10-500 [micro]m whereas the TCP scaffolds (85 vol.% porosity) have been fabricated via direct foam casting method with layered microstructure having pore sizes above 500 [micro]m. Both the scaffolds after sterilization and coating with 0.1% human serum have been used for cell culture studies. The interaction between human bone marrow derived [CD34.sup.+] HSCs and the bioceramic and Ti scaffolds has been studied in the presence of absolutely serum free hematopoietic progenitor growth medium. The results have shown that in the presence of serum free medium, the HSCs attach to both Ti and bioceramic scaffolds and the adhesion percentage of HSCs significantly increased with the incubation time on the bioceramic scaffold compared to that on titanium scaffold.
Subject: Titanium
Hematopoietic stem cells
Porosity
Phosphates
Metal powder products
Metal powders
Authors: Rajyalakshmi, A.
Balasubramanian, K.
Mishra, Sarika
Pub Date: 08/01/2010
Publication: Name: Trends in Biomaterials and Artificial Organs Publisher: Society for Biomaterials and Artificial Organs Audience: Academic Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2010 Society for Biomaterials and Artificial Organs ISSN: 0971-1198
Issue: Date: August, 2010 Source Volume: 24 Source Issue: 2
Product: Product Code: 3399180 Powdered Metal Parts; 3399100 Metal Powders & Paste NAICS Code: 332117 Powder Metallurgy Part Manufacturing; 33149 Nonferrous Metal (except Copper and Aluminum) Rolling, Drawing, Extruding, and Alloying SIC Code: 3499 Fabricated metal products, not elsewhere classified; 3399 Primary metal products, not elsewhere classified
Accession Number: 308129447
Full Text: Introduction

HSCs are very well known for their self renewal capacity and are clinically used in transplantation therapy of various hematological disorders. In spite of the phenomenally huge literature available in the field [1-3] numerous challenges still remain for successful manipulation of Bone Marrow Hematopoietic Stem Cells (BM-HSCs) for regenerative therapies. Therefore, many investigations are currently undertaken to study the function of microenvironment signals of the hematopoietic stem cell (HSC) niche for better control of transplanted HSCs [4]. Recently, two possible inductive microenvironments have been proposed: an osteoblastic niche where BM-HSCs interact with osteoblasts; a second niche called the vascular niche is associated with endothelium where stromal cells including endothelial cells interact with BM-HSCs [4-7]. Many studies indicate that BM-HSCs self-renewal and homing are adhesion dependent. Not only local growth factors and direct cell-cell interaction are thought to control the balance between BM-HSCs expansion and differentiation, but also the composition of the extra-celluar matrix (ECM) is found to play an important role in providing specific adhesion characteristics between BM-HSCs and the surrounding bio-macromolecules.

Interaction of HSCs with their specific microenvironments, known as stem cell niches, is critical for maintaining stem cell properties, including self-renewal capacity and the ability to differentiate into multiple lineages. The 3D architecture of internal microenvironment of the bone marrow along with the rich milieu of ECM proteins plays an integral part in signaling control of BM-HSCs migration, proliferation and differentiation. It appears that both substrate topographical and biochemical cues promote BM-HSCs adhesive behaviors, which are crucial for Bone marrow-derived hematopoietic stem cells (BM-HSCs) homing, self-renewal and lineage commitment within their microenvironment [15, 16].

Due to the non-adherent property of BM-HSCs on tissue culture polystyrene (TCPS), BM-HSCs are conventionally cultured in suspension within special spinner flasks or bioreactors in a dynamic culture [10]. However, this culture system failed to maintain cell localization to specific environment for close cell-cell and cell-matrix interaction [10]. So BM-HSCs have to proliferated and differentiated by providing suitable three-dimensional (3D) niche-like microenvironments [20, 21] to mimic the natural scenario using matrix and tissue engineering approaches. Additional evidence supports a role for the ability of the osteoblastic niche to retain stem cells in a quiescent state is an important mechanism in maintaining sufficient stem cells in the bone marrow environment [22]. Bone tissue regeneration in vivo on porous titanium and bioceramic scaffolds involves maintenance and expansion of HSCs since the osteoblastic niche itself supports and maintains HSCs [12-13].Therefore, this study is of great interest, as it can lead to valuable insights and understanding variable considerations to get efficient hematopoiesis in vivo at the site of bone reconstruction [22]. The primary goal of this study was to visualize the adhesion pattern of HSCs on two different types of scaffolds with a reference of Tissue Culture Polystyrene (TCPS). Indeed, the aim of using scaffolds coated with human serum was to further increase the adhesion efficiency.

Materials and Methods

Raw Materials and Reagents

Commercially pure titanium grade 2 and tricalcium phosphate (s. d. fine chemicals ltd.) were used as scaffolding materials. Dulbecco's Minimal Essential Medium (DMEM), Dulbecco's Minimal Essential Medium F-12(DMEM F-12), Dulbecco's Phosphate Buffered Saline (DPBS), Fetal Bovine Serum, Penicillin (100U/ml), Streptomycin (100 ig/ml), 200 mM L--Glutamine, Trypsin-EDTA (0.25%), Trypan Blue were procured from GIBCO Invitrogen. Ficoll (1.077-1.080 g/ml at 20%C) (0.5%) was procured from sigma chemicals ltd. Hematopoietic Growth Progenitor Medium (HGPM) and Iscove's Modified Dulbecco's Medium (IMDM) were procured from Lonza.

Human cells, recombinant cytokines, and culture medium

Frozen human [CD34.sup.+] cells were purchased from (Lonza) which were obtained from normal volunteers participating in an approved donor program (Lonza). Cells (92% [CD34.sup.+], 95% viable) were thawed according to the reference. The recombinant human stem cell factor (SCF), Flt-3 ligand (FL), thrombopoietin (TPO), interleukin-3 (IL-3), StemSpan Medium, MethoCult GF+ H4435, and MyeloCult H5100 were all from Stem Cell Technologies (Vancouver, BC, Canada).

Preparation, Cleaning and Sterilization of the Scaffolds

Titanium (Ti) scaffolds [Fig. 1 (a)] were prepared through powder metallurgy route. The composition for titanium scaffolds consist of commercially pure titanium grade 2 (0.2% Fe, 0.18% O, 0.05% N, 0.06% C, 0.013% H), which is currently used in dental and orthopaedic implants. The diameter of the Ti scaffold was kept as 18.7 mm with a height of 2.9 mm whereas the diameter and height of bioceramic scaffold were 17 mm and 6.7 mm respectively. The scaffolds were manufactured by standardized methods. Before insertion into the cell culture, the titanium scaffolds were rinsed with distilled water, mechanically cleaned in ultrasonic baths with ethanol and double distilled water and incubated for 10 min in 65% HN[O.sub.3] in order to remove organic and inorganic residues [18]. The ultrasonicated scaffold was further characterized for its microstructure through SEM. Finally, after rinsing with distilled water, and drying in dust free air, the scaffolds were further sterilized for 15 min (121[degrees]C, 15 bar pressure) by autoclaving. Bioceramic (TCP) scaffolds (Fig. 1 (b)) were prepared through direct foam casting route, sintered immediately at 1295[degrees]C and characterized for their density and microstructure. These scaffolds were further machined, grinded and shaped for further in vitro analysis and cell viability tests. For cleaning, the ceramic scaffolds were rinsed with distilled water and mechanically cleaned in ultrasonic baths with double distilled water and further autoclaved after drying in dust free air. The clean titanium as well as bioceramic scaffolds were sterilized in autoclave and made ready for inoculation. Both the scaffolds after sterilization have been used for cell culture studies.

[FIGURE 1 OMITTED]

Cell Culture

One thousand thawed CB [CD34.sup.+] cells were added to 100 iL Stem Span serum-free medium (1% BSA, 10 ig/mL recombinant human insulin, 200 ig/mL human transferrin, [10".sup.4]M 2-mercaptoethanol and 2 mM L-glutamine in Iscove's Modified Dulbecco's medium) at 37[degrees]C for 10 days supplemented with 40 ig/mL low density lipoprotein and a combination of cytokines: 100 ng/mL Stem Cell Factor, 100 ng/ mL FL, 50 ng/mL TPO, and 20 ng/mL IL-3. Cells were maintained in a 5% C[O.sub.2] humidified incubator at 37 [degrees]C for 24hrs.

Seeding of the HSCs into the Scaffolds

Porous Titanium scaffold or bioceramic scaffold was placed at the bottom of the well in a 24-well cell culture plate (Corning). Each well has an internal diameter of 19 mm. The scaffolds were coated with human serum before seeding of HSCs. The human serum (0.1%) screened for viruses and mycoplasma is used for coating the scaffolds. The scaffolds were dip coated once with the human serum and dried in C[O.sub.2] incubator for 24 hrs at 37[degrees]C. Tissue culture polystyrene (TCPS) dish/well was used as a control substrate. One thousand cells were seeded to scaffold by pipetting one ml of cell suspension in the serum free medium supplemented with one ml of Hematopoietic progenitor growth medium (HPGM) directly onto the top of the scaffold slowly. The cell culture plate was incubated at 37[degrees]C and 5% C[O.sub.2] (Shell Lab C[O.sub.2] Incubator) continuously for 10 days. The culture plates were monitored and the medium was changed after every 72 hrs. On day 10, cells were harvested by repeated pipetting and counted on a hemocytometer. Each culture with a single material was prepared in duplicates or triplicates.

Scanning Electron Microscopy (SEM) of the Scaffolds

The seeded scaffolds were fixed in 2% glutaraldehyde in DPBS, dehydrated over an ascending propanol concentration, critical point dried (in-house designed system) and sputter coated with gold sputter coater (Polaron range, SC7620). The specimen were mounted onto stubs with silver paint and examined in a Scanning Electron Microscope (TESCAN VEGA LSU, Detector Oxford Inst.)

Statistical Analysis

Values at least triplicate were averaged and expressed as mean [+ or -] standard deviation (SD). Each experiment was repeated three times and the significance was calculated using students t-test. Differences were considered statistically significant at p < 0.05.

[FIGURE 2 OMITTED]

Results and Discussion

Morphology of Ti and Bioceramic Scaffolds

The Ti scaffolds (20 vol. % porosity) have shown the pore size distribution in the range of 10-50[micro]m [Fig. 2 (a)] whereas the TCP scaffolds (45 vol. % porosity) had layered microstructure with pore sizes above 200|m (Fig 2 (b)).

Adhesion patterns of HSCs on the Scaffolds

The results have shown that in the presence of serum free medium, the HSCs tend to attach to the periphery of the Ti scaffold whereas in the case of bioceramic scaffold, they tend to attach all over the material. The comparison of adhesion patterns of the HSCs on various substrates at different time points in the serum free medium is shown in Fig. 3. It was found that Tissue Culture Polystyrene (TCPS) in purple bars exhibited the lowest capacity (5.01%) for cell adhesion even after 60 min of incubation. This is consistent with the non-adherent property of HSCs on TCPS. In contrast, Ti and Bioceramic scaffolds showed an increased adherence of 16.7% and 32.7% respectively for HSCs in serum free medium.

[FIGURE 3 OMITTED]

The results suggest that the surface topography of Ti and bioceramic material facilitated HSCs attachment (Fig. 4 & 5). Among them, pure TCP bioceramic scaffold which was coated with human serum displayed the highest degree of cell adhesion (Fig. 4). However, its application in tissue engineering [23] is limited by the rapid absorption rate and weak mechanical strength. In contrast, the Ti scaffolds on which the adhesion of HSCs was in-between have better potential to carry HSCs as they are immensely useful in the tissue engineering of implants with load bearing applications [Fig 5 (a) and (b)]. However, in order to further increase its adhesion efficiency, human serum was physically coated onto the surface of Ti scaffold.

[FIGURE 4 OMITTED]

In this study Ti and bioceramic scaffolds for adhesion of HSCs have been fabricated. Although the powder metallurgy and direct foam casting routes have been explored from decades, yet they provide simple and rapid methods for fabricating Ti and bioceramic scaffolds. It is also possible to use other methods including biochemical modifications [19] and chemical grafting [12] to introduce extra cellular matrix (ECM) proteins onto the substrate surface. However, one of the problems with these techniques is the slow mass transfer of proteins into 3D porous matrix. Coating of the serum proteins on the Ti and bioceramic surfaces provides efficient and cost effective solution to carry HSCs and also uses smaller amounts of serum for protein immobilization.

[FIGURE 5 OMITTED]

Serum free medium was used because serum in the culture may lead to undefined differentiation of the HSCs. Whereas extremely small concentration of high purity screened human serum was used for coating the scaffolds so that it merely acts as a physical coating (after it is dried) for enhanced adhesion. The existence of ECM molecules produced by the cells on the surface of Ti scaffolds and in the interior of the porous bioceramic provides sustained cell recognition signals even though TCP is bioresorbable.

Cues emanating from the HSC niche play a crucial role in regulating HSCs self-renewal and differentiation in vivo [8-9]. The frequent and complex signal exchanges among BM-HSCs and other cells (osteoblasts and stromal cells such as endothelial cells, fibroblasts) as well as intimate interactions with ECM components in this niche are enabled by the close contact within this microenvironment [12, 17]. However, the conventional HSC suspension culture is characterized by non-adherent HSCs dispersed in large amounts of aqueous culture medium within a dynamic spinner flask or a stirred bioreactor [10]. This artificial setting leads to the dilution of signaling molecules excreted by the cells. The suspended cells also have no interactions with an anchoring surface such as ECM in a natural microenvironment. The lack of cell-to-cell and cell-to-matrix interaction is one of the factors that are responsible for the failure of maintaining self-renewal and differentiation abilities of HSCs during in vitro culture [4, 10, 13-14].

A bioceramic scaffold coated with serum proteins which would mimic the micro environment provided by the bone marrow niche is constructed in this study. Incorporation of the natural serum proteins in the scaffold and effective adhesion of HSCs by the scaffolds allows for close cell-matrix interaction and cell-cell interactions. Therefore, a matrix with effective adhesion of HSCs can also provide the possibility of housing cells at a higher density. The proliferation and differentiation of HSCs within such a construct can be further enhanced by incorporating the necessary and desirable co-cultured cells and critical growth factors and cytokines.

Conclusion

In the present study, a serum coated bioceramic scaffold fabricated by direct foam casting technique was introduced for adhesion of HSCs. The results indicate the feasibility of promoting the adhesive characteristic of HSCs by modulating the topographical and biochemical properties of the culture substrate. This study suggests the great potential for designing bio-mimetic artificial carriers as efficient anchorage systems for BM-HSCs to facilitate cell expansion or differentiation functions.

Acknowledgements

We thank Dr. V.S. Raghunathan for his expert advice and assistance in scanning electron microscopy. We also thank Dr. Lakshmi Kiran and Dr. K.S. Ratnakar from Global Clinical Reference Laboratories, Hyderabad for their support in collection of peripheral blood and bone marrow samples.

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A. Rajyalakshmi, K. Balasubramanian and Sarika Mishra *

Non-Ferrous Materials Technology Development Centre, P.O. Kanchanbagh, Hyderabad-500058, INDIA

Corresponding Author: uptecsarika@rediffmail.com (Sarika Mishra)

Received 24 December 2009, Accepted 27 April 2010, Published online 18 August 2010.
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