Recruitment of Endogenous Stem Cells for Tissue Repair

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Recruitment of Endogenous Stem Cells forTissue Repair

Jing Zhao, Ning Zhang, Glenn D. Prestwich, Xuejun Wen*

J. Zhao, N. Zhang, X. WenDepartment of Bioengineering, Clemson University, Clemson, SC29634, USAE-mail: [email protected]. Zhang, X. WenDepartment of Cell Biology and Anatomy, Orthopedic Surgery,Medical University of South Carolina, Charleston, SC 29425, USAG. D. PrestwichDepartment of Medicinal Chemistry and the Center for Thera-peutic Biomaterials, University of Utah, 419 Wakara Way,Suite 205, Salt Lake City, UT 84108, USA

The traditional concept of stem cell therapy envisions the isolation of stem cells from patients,propagation and differentiation in vitro, and subsequent re-injection of autologous cells intothe patient. There are many problems associated with this paradigm, particularly during thein vitro manipulation process and the delivery and local retention of re-injected cells. Analternative paradigm that could be easier, safer, and more efficient, would involve attractingendogenous stem cells and precursor cells to the defect site for new tissue regeneration.Hepatocyte growth factor (HGF), a pleiotropic cytokine of mesenchymal origin, exerts a strongchemoattractive effect on mesenchymal stem cells (MSCs) and neural stem cells (NSCs), andinduces migration of MSCs in vitro. However, HGF undergoes rapid proteolysis in vivo, whichresults in a very short lifetime of the bioactive cytokine. To maintain the therapeutic level ofHGF at the defect site necessary for endogenous stem cell recruitment, sustained, long-term,and localized delivery of HGF is required. Thiol-modified glycosaminoglycans hyaluronan (HA)and heparin (HP), combined with modified gelatin (Gtn), have been crosslinked withpoly(ethylene glycol) diacrylate (PEGDA) to afford semisynthetic ECM-like (sECM) hydrogelsthat can both provide controlledgrowth factor release and permit cellinfiltration and proliferation. Hereinwe compare the use of different sECMcompositions for controlled release ofHGF and concomitant recruitment ofhuman bone marrow MSCs into thescaffold in vitro.

Macromol. Biosci. 2008, 8, 836–842

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Introduction

The traditional concept of cell therapy is based upon

several basic steps. The first step is cell sourcing and cells

may be isolated from autologous, allogenic, or xenogen-

icsources. Second, the isolated cells are expanded in vitro to

a cell population sufficient for effective treatment. The

expanded cells can also be seeded on a scaffold and

cultured in a bioreactor. Finally, the expanded cells are

re-implanted into the patient. However, this final process

is associated with ethical, economic, regulatory, and

clinical problems.

DOI: 10.1002/mabi.200700334

Recruitment of Endogenous Stem Cells for Tissue Repair

Clinically, allogenic and xenogenic sources face the

greatest likelihood if immune rejection by the patient.

Ethical and regulatory issues must also be resolved for this

to be a routine clinical treatment. Thus, autologous cells

would seem to be the best choice, but cell isolation from

patients in need of treatment can cause additional normal

tissue morbidity. In order to obtain sufficient numbers

for transplantation, in vitro proliferation is essential,

which may cause undesirable phenotype change.[1] The

pluripotency of stem cells may decrease during in vitro

culture.[2,3] Allogenic and xenogenic components used in

culture may cause host immune rejection. In addition,

the cost of in vitro expansion of stem cells is very high,

since a battery of growth factors is needed for the

propagation procedures. The economic and multi-week

expansion period present important challenges to these

clinical procedures. Finally, while mesenchymal stem cells

(MSCs) attract much attention because of their pluripo-

tency, the pluripotency of MSCs decreases during in vitro

culture[2,3] using conventional two-dimensional (2D)

culture conditions.

An alternate cell source could be endogenous stem cells.

Indeed, a regenerative medicine approach for tissue repair

focused on the direct manipulation of endogenous adult

stem cells is very appealing. There are several advantages

to the use of endogenous stem cells for tissue repair. First,

using endogenous stem cells avoids the immunocompat-

ibility issues that accompany the use of allogenic and

xenogenic cells. Second, it is easier, safer, and more

efficient to use endogenous stem cells for tissue repair to

expand and re-implant autologous cells. Third, only a

single surgical intervention is required, rather than two

surgeries several weeks apart. Finally, the process of

recruiting endogenous stem cells offers both regulatory

and economic advantages relative to ex vivo approaches.

The utilization of endogenous stem cells may be

enhanced in two ways. One strategy is to mobilize the

endogenous stem cells into the circulation. For example, it

is reported that granulocyte colony-stimulating factor

(G-CSF) mobilizes stem and progenitor cells from the bone

marrow into the peripheral blood, from which they can

‘home’ into the lesion site in the brain and have a

protective or restorative effect.[4] Also, the mobilized

endogenous stem cells are showing promising outcomes

for cardiac repair.[5] A second strategy is to enhance the

recruitment of endogenous stem cells into the lesion

site for tissue regeneration. Several factors, such as

hepatocyte growth factor (HGF)[6] and stromal cell-derived

factor-1 (SDF-1)[7] have shown chemotaxic effects on

stem cells. In this study, we used HGF as a model factor

to attract bone marrow MSCs into an extracellular matrix

(ECM)-like hydrogel in vitro. Hepatocyte growth factor

(HGF) has been shown to be a strong chemotactic factor for

the mobilization and migration of MSCs.[6] Exposure of



MSCs to HGF inducedmigration of MSCs in vitro. However,

like most proteins, HGF undergoes rapid proteolysis

in vivo, which results in a very short lifetime of the

bioactive growth factor. The half-life of HGF that was

delivered in a soluble form in vivo is 3–5 min. In contrast,

the time required to recruit a sufficient number of stem

cells for tissue repair is usually days to weeks. Therefore,

direct injection of growth factors to the repair site has

limited success.

To maintain the therapeutic level of HGF at the repair

site necessary for endogenous stem cell recruitment,

sustained, long-term, and localized delivery of HGF is

essential. Several polymer delivery systems are being

developed for proteins and growth factor delivery.

Reservoir devices, solid implants, polymeric micro- and

nanoparticles, and hydrogels are themost commonly used.

Polymer systems have many advantages; for example,

they can stabilize proteins, provide localized delivery, and

produce diffusion-limited concentration gradients in

tissues. ECM-derived polymers with a wide array of

physiological functions represent ideal substrates for

HGF delivery and stem cell recruitment, since ECM based

materials may provide adhesion sites for migrating stem

cells to grow in. Recently, an injectable, in situ-

crosslinkable semisynthetic ECM-like hydrogel, or sECM,

was created by crosslinking thiol-modified hyaluronan

(HA) and gelatin (Gtn) using poly(ethylene glycol)

diacrylate (PEGDA).[8] This material offers a flexible

composition and compliance, simplicity of use, and was

developed for ease of translation from in vitro to preclinical

in vivo as well as clinical uses.[9] In this study, we

compared different compositions of the sECM hydrogel for

controlled release of HGF to recruit human bone marrow

mesenchymal stem cells (hMSCs) to the scaffold in vitro.

Experimental Part

Materials

Thiolated chemically modified HA (CMHA-S), thiol-modified

gelatin (Gtn-DTPH), thiol-modified heparin (HP-DTPH), and PEGDA

were kindly provided by Glycosan BioSystems Inc. (Salt Lake City,

UT). Bovine serum albumin (BSA) and heparin were purchased

from Sigma Chemical Co. (St. Louis, MO). Human hepatocyte

growth factor (HGF) was purchased from PeproTech (Rock Hill, NJ).

HGF ELISA kit was purchased from Biosource International, Inc.

(Camarillo, CA). Transwells of 8 mmpore size were purchased from

Corning Costar Inc. (Corning, NY). Human bone marrow-derived

mesenchymal stem cells (hMSCs) were obtained from Lonza

(Allendale, NJ). The expression of CD105 and CD44 was

characterized before use for this study. The expression of CD105

and CD44 was more than 92%. hMSCs were maintained in

culture in MSCGM–Mesenchymal Stem Cell Medium (Lonza,

Allendale, NJ).

www.mbs-journal.de 837

J. Zhao, N. Zhang, G. D. Prestwich, X. Wen

838

Hydrogel Preparation for in vitro Experiments

Two different sets of hydrogels were prepared. For HA:Gtn gels,

CMHA-S and Gtn-DTPH in DI water solution (pH 7.4) were mixed

in a volume ratio of 1:1, and then the HA:Gtn solution was

cross-linked by mixing it with 2 wt.-% PEGDA in deionised (DI)

water in a volume ration of 4:1. The final concentration of both

CMHA-S and Gtn-DTPH was 0.4% w/v. For HA:Gtn:HP gels,

HP-DTPH in DIwater (pH 7.4) wasmixedwith the HA:Gtn solution

mentioned above: the concentration of HPwas 0.3%w/v. HGFwas

incorporated non-covalently into the gels by pre-mixing 500 ng of

HGF with 200 mL of the modified HA and gelatin solution prior to

cross-linking. Gels formed within 20 min.

In vitro Release Kinetics of HGF

HGF was allowed to release from these hydrogels at 378C into a

phosphate buffered saline (PBS) solution supplemented with 1%

BSA, 1� 10�3M EDTA, and 10mg �mL�1 of heparin. At days 1, 2, 3, 5,

9, 14, 20, and 26, 100 mL of conditioned

medium was sampled, and an equal volume

of fresh release medium was added back to

maintain the total volume. Samples were

then immediately frozen at �20 8C until

measurement. Each release condition was

performed in triplicate.

HGF Measurement with Sandwich

Enzyme-Linked Immunosorbent

Assay (ELISA)

The amount of HGF released was quantified

by a sandwich enzyme-linked immunosor-

bent assay (ELISA) using an assay from a

commercially available kit (Biosource). Briefly,

100 mL of the sample was added into the

designated ELISA wells and incubated at 4 8Covernight. After the incubation, the plate was

washed four times with a wash buffer. Biotin

antibody (100 mL) was then added into each

well, and the plate was incubated at room

temperature for 1 h. The plate was then

washed four times, and 100 mL of Streptavidin

solution was added and incubated for 45 min

at room temperature. The substrate was

added to the wells and the absorbance was

measured at 450 nm. The total accumulated

release of HGF was calculated by integration

of the individual measurements over the

cumulative time of the experiment.

Figure 1. Cumulative in vitro HGF release from HA:Gtn and HA:Gtn:HP hydrogels. A)Release amount in nanograms and B) percentage of release.

Cell Migration Assay

The migration capacity of hMSCs was ana-

lyzed using Costar Transwell invasion cham-

bers with polycarbonate membrane filters of

6.5 mm diameter and 8 mm pore size to form



dual compartments in a 24-well tissue culture plate. Samples each

containing 104 cells in 200 mL of media were added to the upper

compartments. The lower compartments were covered with

200 mL of hydrogel, with or without HGF as a source of

chemoattractants, and 400 mL of culture media were added. The

migration chambers were incubated for 8 h at 37 8C and 5% CO2.

After incubation, cells on the top surface of the filters were wiped

off with cotton swabs. Cells that had migrated into the lower

compartment and attached to the lower surface of the filter were

counted after being stained with Alexa 546-phalloidin and

Draq 5. After being fixedwith 4% formaldehyde solution in PBS for

10 min at room temperature, cells were permeabilized using

0.1% Triton X-100 in PBS for 5 min and washed three times

with PBS. Alexa 546-phalloidin (1.5�10�6M) solution in PBS

was added into the samples for 20 min at room temperature.

To reduce non-specific background staining, 1% BSA was added to

the staining solution. The samples were washed three times with

PBS.

DOI: 10.1002/mabi.200700334


The number of cells that penetrated into the hydrogel was

counted by taking pictures using a laser confocal microscope

(Leica) for 100 mm thickness. The total number of cells at that

thickness was counted using Image Pro Plus.

Results and Discussion

Cumulative in vitro HGF Release

Immobilization of heparin in an sECM has been demon-

strated to protect growth factors from enzymatic degrada-

tion and thermal denaturation.[10] These crosslinked

heparin-containing sECMs behave in an analogous fashion

to a heparan sulfate proteoglycan, extending the release

times of growth factors in vitro and in vivowhile retaining

their bioactivity.[11,12] The effect of immobilized heparin

on the controlled release of HGF from sECM hydrogels was

measured in vitro. Figure 1 presents the time course of

total cumulative release for HGF. Covalently cross-linked

heparin slows down the HGF release significantly in HA:

Gtn based gels, but also reduces the net quantity released

at a given time point. Without heparin, HA:Gtn gels

released a total of 35% of the initially loaded HGF in 26 d.

In contrast, HA:Gtn:HP gels released a total of 18% of their

loaded HGF at the end of 26 d.

Figure 2. An 8 mm pore size transwell.

Bone Marrow Mesenchymal Stem Cells Migration

In this experiment, four groups were assigned. In the

negative control group, no HGF was present in either

the lower or upper compartment of Transwells. In the

positive control group, 20 ng �mL�1 of HGF was present in

the culture media of the lower compartment of Transwell.

In the third group, 500 ng of HGF encapsulated in a HA:Gtn

gel was placed in each lower compartment. In the last

group, 500 ng of HGF encapsulated in a HA:Gtn:HP gel was

placed in each lower compartment.

In order to analyze the migration ability of hMSCs in

response to released HGF, two locations were observed

under a confocal microscope: the lower surface of the filter

and the hydrogel in each lower compartment (Figure 2).

Figure 3 shows the number of MSCs that migrated to the

lower surface of the filter. All four groups (Figure 3B–D)

showed significantly greater number of MSCs on the lower

surface of the filter than the negative control group

(Figure 3A). Figure 4 presents the cell density in cells

per mm2 on the lower surface of the filter. Significantly

more MSCs migrated across the filter than for each group

relative to the negative control.

The number of cells recruited into the sECM gels in the

lower compartments were also determined. Large num-

bers of cells were found inside the HA:Gtn gel loaded with

500 ng of HGF (Figure 5C). Indeed, significantly more cells



were found in the HA:Gtn gel loaded with 500 ng of HGF

(Figure 5C) than in any of the other three groups: the

negative control group without HGF loading (Figure 5A),

the positive control groupwith 20 ng �mL�1 of soluble HGF

inmedium (Figure 5B), and the HA:Gtn:HP hydrogel loaded

with 500 ng of HGF (Figure 5D). Paradoxically, no cells were

found in the HA:Gtn:HP gel loaded with 500 ng of HGF

(Figure 5D), similar to the cell density in the negative

control group Figure 5A. In the heparin-DTPH containing

hydrogel, the HGF release rate is about two times slower

than the gel without heparin-DTPH. This slow release may

prevent the strong HGF gradient formation. In the culture

media, the HGF amount is lower in the HP-DTPH

containing gel than in the HA:Gtn gel. Therefore, hMSCs

will sense the HGF and migrate across the membrane.

However, the HGF gradient is not high enough to attract

hMSCs to migrate into the hydrogels.

General Discussion

Thiolated hyaluronan is biocompatible and can be adapted

to produce a variety of desirable biologic effects.[13] Gelatin

is a partially hydrolyzed collagen product, which is an

excellent substrate for cell attachment, proliferation, and

differentiation. Both thiolated HA[14,15] and thiolated

gelatin[16,17] have been used in a number of tissue

engineering applications, including the delivery of basic

fibroblast growth factor (bFGF) for cutaneous wound

repair[18] and human demineralized bone matrix for bone

repair.[13] The sECM created by co-crosslinking thiol-

modified HA with thiol-modified gelatin produces

mechanically robust, bioresorbable scaffolds that can be

implanted to achieve tissue growth in vivo.[13,19–21] The

sECM is also an effective vehicle for cell delivery and

retention in vivo, including the repair of osteochondral

defects using encapsulated autologous MSCs.[13] Growth

factors delivered locally using the sECM hydrogels elicit a

strong angiogenic response, even when the growth factors

are delivered in low, nanogram doses.[11–13,22] In addition,

it is reported that incorporation of small amounts of

heparin in the gels can prolong and spatiotemporally



Figure 3. MSCs that have crossed the 8 mm pores to the lower surface of the filter. A)Negative control (no HGF), B) positive control (20 ng �mL�1 of HGF in culture media), C)500 ng of HGF encapsulated in a HA:Gtn hydrogel, D) 500 ng of HGF encapsulated in aHA:Gtn:HP hydrogel, and E) 1 000 ng of HGF encapsulated in a HA:Gtn:HP hydrogel.

840

regulate the rate of growth factor release in vitro.[11,12] In

this study, we demonstrated that through delivery of HGF

from heparin-containing HA:Gtn hydrogels or HA:Gtn

hydrogel only, the biologic activity of HGF can be

maintained, and the human MSCs are most effectively

recruited into the HGF releasing hydrogels that lack the

immobilized heparin component.

Figure 4. The number of MSCs that have crossed the 8 mm pores to the lower surface o



As commonly occurs with highly

bioactive polypeptides in vivo, HGF

undergoes rapid proteolysis and has an

in vivo half-life of 3–5 min. Figure 1

illustrates that release of HGF from

crosslinked thiol-modified HA and gela-

tin hydrogel in vitro could be sustained

for up to 3 to 6 months based on the

extrapolated value from a 26-day study.

This is a dramatic increase in time of

availability compared to the short half-

life of free HGF in vivo. Furthermore, the

rate of HGF release could be controlled

with heparin. In the absence of heparin,

HGF was released twice as fast from the

HA:Gtn hydrogel than the HA:Gtn:HP

hydrogel. This result is consistent with

extended release of bioactive keratino-

cyte growth factor (KGF), vascular

endothelial growth factor (VEGF), angio-

poetin-1 (Ang-1), and bFGF from similar

heparan-sulfate mimetic sECM hydro-

gels.[10–12]

The biological activity of the HGF

released from the hydrogels was con-

firmed by in vitro hMSC migration experiments. On the

lower surface of each filter, all groups except the negative

controls showed an obvious accumulation of cells (Figure 3

and 4). In the hydrogels in the lower compartments, many

cells migrated into the HA:Gtn gel loaded with 500 ng of

HGF, while there were no cells attached on the negative

control group or the HA:Gtn:HP gel loaded with 500 ng of

f the filter.

DOI: 10.1002/mabi.200700334


Figure 5.MSCs that havemigrate into the hydrogels on the lower compartment. A) Negativecontrol (no HGF), B) positive control (20 ng �mL�1 of HGF in culture media), C) 500 ng of HGFencapsulated in a HA:Gtn hydrogel, D) 500 ng of HGF encapsulated in a HA:Gtn:HP hydrogel,and E) 1 000 ng of HGF encapsulated in a HA:Gtn:HP hydrogel.

HGF (Figure 5 and 6). This seemingly paradoxical result

seems to indicate that the amount of loading and the

release rate of HGF from the gel are both crucial

parameters that affect the migration of hMSCs. If the

release rate or amount of HGF is lower than the threshold

for hMSC response, there will be no control of hMSCs

migration, since when we double the amount of HGF

loading into the HA:Gtn:HP gel to 1000 ng (Figure 5E), a

Figure 6. The number of MSCs that have migrated into the hydrogels on the lower compart



significant amount of hMSCs mi-

grated into the HA:Gtn:HP hydrogels.

The other interesting indication is

the importance of the HGF gradient

in the control ofMSCmigration. If we

compare the 20 ng �mL�1 HGF group

(Figure 5B) with the 500 ng �mL�1

group (Figure 5C), fewer MSCs are

migrated to the hydrogel in group II

than that in group III. There is a

sufficient amount of HGF in group II

for MSCs to migrate, however, the

difference between group II and III is

the establishment of a HGF gradient.

A HGF gradient is well established in

group III as a result of the constant,

slow release of HGF from the sECM

gel.

Conclusion

The release characteristics of HGF

and stem cell recruitment capacity of

a series of sECM hydrogels loaded

with HGF have been investigated

in vitro. These sECM hydrogels provide sustained release of

biologically active HGF in vitro, with HGF release sustained

for over three weeks. This is a dramatic increase in time of

availability compared to the short half-life of free HGF

in vivo. HGF released from a HA:Gtn sECM hydrogel

attracted human bone marrow MSCs to migrate into the

hydrogel following the HGF gradient established by the

HGF-loaded HA:Gtn hydrogels.

ment.



842

Acknowledgements: Financial support for this project wasprovided by NSF (No. 0132573 to XW and the Utah Centers ofExcellence Program to GDP). We thankGlycosan Biosystems Inc. fora gift of Extracel components of the sECM.

Received: December 18, 2007; Revised: March 29, 2008; Accepted:April 1, 2008; DOI: 10.1002/mabi.200700334

Keywords: biomaterials; endogenous stem cells; hepatocytegrowth factor (HGF); hyaluronan (HA); hydrogels

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Recruitment of Endogenous Stem Cells for Tissue Repair