IN VIVO AND IN VITRO CHONDROGENESIS OF ADULT BONE MARROW-DERIVED MESENCHYMAL STEM
CELLS IN A GELATIN SCAFFOLD
+Gao, J; Dennis, JE; *Goldberg, VM; Caplan, AI
Skeletal Research Center, Department of Biology and *Orthopaedics, Case Western Reserve University, Cleveland, Ohio
Introduction
Purified and culture-expanded adult bone marrow-derived mesenchymal
stem cells (MSCs) have the potential to differentiate into multiple
mesenchymal lineages including bone, cartilage, and tendon. A
commonly used methodology to evaluate in vitro chondrogenesis of
MSCs is a micro-mass culture, in which the culture-expanded MSCs are
centrifuged into a pellet that is then incubated in a chemically defined
medium supplemented with growth factors (1). Using tissue engineering
principles, these MSCs were combined with different cell-carriers to
repair cartilage defects. However, the events MSC progression to
cartilage in a scaffold have not been thoroughly investigated. This
information is useful to improve the approaches for engineering a
cartilage tissue. The purpose of this study was to characterize the
temporal and sequential cellular and molecular events of in vitro
chondrogenesis of hMSCs in a collagen matrix, and the in vivo
chondrogenesis of this tissue engineered construct was also observed.
Materials and Methods
Cell culture: Human bone marrow harvesting, MSC isolation, and
culture expansion were accomplished following a standard protocol
previously published [2]. Cells at the first passage were used for all
experiments. In total, 8 individual MSC preparations were used in this
study.
Cell loading into the scaffold: Discs of gelatin sponge (Gelfoam ,
Pharmacia-Upjohn, Kalamazoo, MI), 4 mm in diameter and 4 mm thick,
were prehydrated with Tyrode salt solution. Immediately before the cellloading, Gelfoam disc was briefly blotted onto a sterile gauze and then
was transferred into a tube containing a cell suspension at a
concentration of 1.0 x 107/ml. A vacuum was applied to enhance the
loading efficiency (3).
Influence of transforming growth factor beta (TGF- ) on the temporal
chondrogenic process: MSC-loaded Gelfoam discs were cultured in a
chondrogenic medium (1) supplemented with TGF- 1 at 0, 2, 4, 8, and
10ng/ml. Gelfoam cartilage discs were separately collected at 1, 3, 5, 7,
and 14 days after culture for histologic and immunohistochemical
analysis.
Morphological studies:
Histology: Specimens were fixed in 10% formalin and embedded
in paraffin. Serial sections of 5 m were cut perpendicular to the surface
of the disc and stained with Toluidine blue.
Immunohistochemistry: Indirect immunoenzymatic assay was
employed to analyze type II and X collagens in the extracellular matrix
(ECM) of the cartilage disc, using monoclonal antibody to type II
collagen and polyclonal antibody to type X collagen.
Morphological observation indicated that the 5ng/ml of TGF- 1 was the
optimal dose for in vitro chondrogenesis. This dose of TGF- 1 was,
therefore, used for following experiments unless otherwise stated.
Subcutaneous implantation: After one week in vitro incubation,
Gelfoam discs were implanted subcutaneously on the dorsal side of the
nude mice in pockets formed by blunt dissection. After 3 weeks,
specimens were recovered for morphological studies.
RNA isolation and RT-PCR analysis of gene expression: Cell-loaded
Gelfoam discs were collected at different time points as stated above.
Total cellular RNA was extracted with a RNeasy Mini Kit (GIAGEN,
Valencia, CA). The isolated RNA samples were reverse-transcripted to
cDNA using random hexamers and Superscript RNase H-Reverse
Transcriptase (SuperScript First-Strand Synthesis system, Life
Technologies, Grand Island, NY), and then amplified by PCR using
gene-specific primers for type II, X collagen, and aggrecan. The
housekeeping gene GAPDH was used as the internal control to monitor
the RNA loading. The RT-PCR products were analyzed by
electrophoresis in a 2% agarose gel.
Results
After cell-loading, MSCs attached onto the fibers of the sponge. The
scaffold began to contract at 48 hours after cell-loading. By 1 week, the
disc contracted to about 55% of its original diameter and about 40 % of
its thickness. No further significant contraction was noted after 1 week.
metachromatic stained ECM was first identified after 7 days in vitro
culture with toluidine blue staining. By 2 weeks, cartilage tissue formed
homogenously in the construct (Fig. 1). Without TGF- , no cartilage
tissue was observed. The dosage of 4 or 6ng/ml of TGF- resulted in
more pores being filled by cartilage through the observation period than
higher (8 or 10 ng/ml) or lower (2 ng/ml) doses. After 3 weeks of
subcutaneous implantation in nude mice, cartilage tissue was observed
to be evenly distributed in the material. The material was mostly
resorbed by this time.
3d
7d
14d
Fig. 1. Histologic pictures of cartilage tissue in the material loaded with
MSCs after 3, 7, and 14 days in vitro culture.
Expression of Type II collagen gene was identified after 3 days of
culture, type X collagen after 6 days, and aggrecan all through the
culture period (Fig. 2)
Fig. 2. Expression of type X, II collagen and aggrecan gene at different
time points of in vitro culture.
Discussion
Chondrogenesis was consistently observed in all 8 tested MSC
preparations when loaded into a gelatin sponge. Our current results are
comparable with the previously reported in which the pellet culture was
used. This tissue engineering approach provides a reliable way to
investigate the cellular and molecular events, such as cell to cell and cell
to matrix interactions, during the MSC chondrogenic process in a tissue
engineering application.
We have observed that the optional dose of TGF- 1 to promote
chondrogenesis in this gelatin sponge was 4 to 6 ng/ml, which is
different from that reported in a pellet culture condition that was
reported as 10ng/ml (1). This discrepancy in TGF- 1 dose may be due to
the contraction of the material after cell-loading. The cell-loaded
Gelfoam disc began to contract at 48 hours after the cell seeding, this
contraction caused 55% reduction in diameter and 40% in thickness by
one week. The contraction reduced the volume of the material and, at the
same time, increased the cell density, which may facilitate cell to cell
and cell to matrix interactions. In vitro and in vivo experiments have
indicated that adequate cell density and cell to cell interactions are
important for chondrogenesis (4).
References
1. Johnstone et al. (1998) Exp. Cell Res. 238:265. 2. Pittenger et al.
(1999) Science, 284:143. 3. Dennis et al. (1992) Cell Transplant. 1:23. 4.
Hall & Miyake: (2000) BioEssays 22:138.
Acknowledgment: This study was supported by grants from NIH.
50th Annual Meeting of the Orthopaedic Research Society
Poster No: 0715