1. Allogenic Human Mesenchymal Stem Cells Seeded on Cortical Cancellous Bone - Paper

7/28/2019 1. Allogenic Human Mesenchymal Stem Cells Seeded on Cortical Cancellous Bone - Paper

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Allogeneic Human Mesenchymal Stem Cells Seeded on

Cortical Cancellous BoneAlloSource

6278 S. Troy Circle, Centennial, CO 80111

AbstractMesenchymal stem cells (MSCs) isolated from cadaveric adipose tissue, can be obtained

in large quantities, and have been reported in the literature to be capable of inducing bone

formation in animal models. In this study, adipose tissue from cadaveric donors was

digested and the resulting stromal vascular fraction (SVF) containing MSCs was seeded onto cortical cancellous bone from the same donor, which had been subjected to a

demineralization process. The resulting MSCs were characterized using flow cytometry

and tri-lineage differentiation (osteogenesis, chondrogenesis and adipogenesis). The finalcell-seeded bone allograft was characterized using histology for microstructure and

biochemical assays for cell count. The resulting grafts have a well-defined cell populationand have the potential to be effective for bone regeneration.

IntroductionMesenchymal stem cells (MSCs) can differentiate along a variety of cell lineages that can

be used to regenerate bone and other tissues[1-4]

. MSCs reside in many tissues including

bone marrow, adipose tissue, synovial fluid, dermis and muscle. Adipose derived MSCsshare many of the characteristics of bone marrow derived stem cells (BMSCs)

[5-11],

including extensive proliferative potential, but are much more abundant and easier to

recover with a higher proliferation rate than BMSCs[12, 13]

. Adipose derived MSCs displayextensive self-renewal capacity to undergo differentiation into many mesenchymal cell

types. Moreover, MSCs have been reported to have low immunogenicity[14-17]. Severalstudies have been reported demonstrating bone regeneration using MSCs from adipose

tissue[12, 13, 18-22]

. These studies have demonstrated that stem cells obtained from adiposetissue exhibit good attachment properties to most material surfaces and have the capacity

to differentiate into osteoblastic-like cells in vitro and in vivo. The objective of this study

is to characterize human MSCs derived from adipose tissue seeded onto corticalcancellous bone, which had been subjected to a demineralization process.

Characterization included: biochemical assay for cell number, flow cytometry and in

vitro tri-lineage differentiation for cell identity, and histology for microstructure.

Materials and Methods

Stem Cell Process

Human cadaveric adipose tissue was recovered from a donor and digested with

collagenase. Cortical cancellous bone was recovered from the same donor and subjected to a demineralization process. The SVF containing MSCs was seeded onto the bone

allografts, after which the non-adherent cells were washed off. The seeded allografts were

put in cryopreservation media and frozen at -80 ºC. The MSCs were characterized using

AlloSource Page 1 M1S0801.00 10/2009



flow cytometry and performed tri-lineage differentiation (osteogenesis, chondrogenesis

and adipogenesis) in vitro. The final grafts were characterized using histology for microstructure and CCK-8 assay for cell count.

MSC Characterization

Flow Cytometry Analysis

The cells were washed with flow cytometric wash buffer (PBS supplemented with 2%

FBS and 0.1% NaN3), stained with the indicated antibodies and washed again before

acquisition. At least 20,000 cells were acquired for each sample on a FACScan flowcytometer (BD Immunocytometry Systems, San Jose, CA). Flow cytometric data were

collected and analyzed using CellQuest software (BD Immunocytometry Systems).

Autofluorescence was assessed by acquiring cells on the flow cytometer withoutincubating with fluorochrome labeled antibodies. Surface antigen expression was

determined with a variety of directly labeled antibodies.

In-vitro Tri-lineage Differentiation

Confluent cultures of MSCs were induced to undergo osteogenesis, adipogenesis and chondrogenesis by replacing the stromal medium with osteogenic, adipogenic and chondrogenic induction medium respectively (Stempro

®differentiation kit, Invitrogen).

Cultures were fed with fresh osteogenic induction medium every 3 to 4 days for a period

of three (3) weeks. Cells were then fixed in 10% neutral buffered formalin. Osteogenicdifferentiation was determined by staining for calcium phosphate with Alizarin Red

(Sigma). Adipogenic differentiation was determined by staining for fat globules with Oil

Red O (Sigma). Chondrogenic differentiation was determined by staining for proteoglycans with Alcian Blue (Sigma).

Final Graft Characterization

Cell count: CCK-8 Assay

Cell Counting Kit 8 (CCK-8, Dojindo Molecular Technologies, Maryland) allows

sensitive colorimetric assays for the determination of the number of viable cells in cell proliferation assays. The amount of the formazan dye generated by the activity of

dehydrogenases in cells is directly proportional to the number of living cells. The final

cell counts were determined from a standard curve based on known numbers of MSCs-only (passage=3).

Histology

When the cultures were terminated, the constructs were cut from the anchors, fixed in

10% neutral buffered formalin (Sigma, St. Louis, MO) for 48 h, put in a processor

(Citadel 2000; Thermo Shandon, Pittsburgh, PA) overnight, and embedded in paraffin.Sections were cut to 5 µm and mounted onto glass slides and stained with hematoxylin

and eosin (H&E). Conventional light microscopy was used to analyze sections for matrix

and cell morphology.




Results

Stem Cell Seeded Grafts

Figure 1 shows pictures of the stem cell seeded grafts: strips and dowels.

Cancellous Top

Cortical Bottom

2 0 mm

50 mm

17mm14mm

Figure 1. Stem cell seeded grafts: str ips and dowels.

MSC Characterization Flow Cytometry

The SVF was stained with CD105, CD90 and CD73 to determine the number of MSCs

present. The immunophenotype of the stromal vascular fraction was consistent from

donor to donor. CD105 was chosen to estimate the mean total percentage of MSCs;

although there is no single surface marker that can discern MSCs in a mixed population.There is a mean total of 7.2% MSCs in the SVF, which is consistent with published

results[23, 24]

.

In-vitro Tri-lineage Differentiation

20X

20X

Negative Control

Al izar in Red Staining Oil Red O Staining Alcian Blue Stain ing

20X

Negative Contro l Negative Contro l

20X A B C

D E F 20X

20X

Figure 2. In-vitro tri-lineage differentiation.




For the osteogenic differentiation, morphological changes appeared during the second

week of the culture. At the end of the 21-day induction period, some calcium crystals

were clearly visible. Cell differentiation was confirmed by Alizarin Red staining (A). The

adipogenic potential was assessed by induction of confluent MSCs. At the end of the

induction cycles (14 days), a consistent cell vacuolation was evident in the induced cells.

Vacuoles brightly stained for fatty acid with Oil Red O staining (B). Chondrogenic

potential was assessed by induction of confluent MSCs. At the end of the induction

cycles (21 days), the induced cells were clearly different from non-induced control cells.

Cell differentiation was confirmed with Alcian Blue staining (C). None of the negative

controls showed any sign of osteogenesis, adipogenesis and chondrogenesis (D,E,F)

respectively.

Final Graft Characterization

Cell count: CCK-8 Assay

28 grafts from 3 donors were tested and shown to have an average of 50,000 ± 13,000live cells/graft.

Histology

H&E was performed to demonstrate cell morphology in relation to the underlying

substrate (cancellous bone matrix). The cells are elongated and adhere to the surface of

the bone substrate.

Figure 3. H&E staining shows cells adhered to the bone substrate.

Cell Nuclei

Bone Substrate




Conclusions and Discussion

Multipotent adult MSCs obtained from cadaveric adipose tissue, have been thoroughly

characterized. The MSCs have been shown to have the capacity to differentiate along

three different lineages. The MSCs have been successfully seeded onto cortical

cancellous bone from the same donor, which has been subjected to a demineralization process. The resulting cell-seeded bone allograft has the potential to be effective for

bone regeneration.

Acknowledgement

AlloSource appreciates the contributions from the following people: Jerry Niedzinski(LABS Inc.), Simon Bogdansky, Jaime Hamil, Adrian Samaniego, Carolyn Barrett,

Jennifer Wesbrook, Kim Gwynn, Brian Dittman, Jason Stephens and Yaling Shi.

Contact Information

Yaling Shi, [email protected]

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1. Allogenic Human Mesenchymal Stem Cells Seeded on Cortical Cancellous Bone - Paper