Abstract
Background: There is no ideal treatment for end-stage degenerative
wrist disorders and subsequent carpal collapse. The purpose of this study was
to investigate whether autologous cartilage constructs tissue-engineered from
bone-marrow-derived mesenchymal stem cells can be effective for carpal bone
reconstruction.
Methods: Total lunate excision was performed in twenty-seven adult
New Zealand White rabbits. Mesenchymal stem cells were isolated from marrow
and then were culture-expanded. Group-1 rabbits underwent excision only.
Group-2 animals underwent excision followed by implantation of a scaffold
consisting of gelatin and hyaluronan. Group-3 animals underwent excision
followed by implantation of a mesenchymal stem cell-seeded scaffold that had
been preincubated in chondrogenic medium. The group-1 animals were killed at
six weeks, whereas the group-2 and group-3 animals were killed at six or
twelve weeks. Tissues were harvested for radiographic and histologic
analysis.
Results: Significant carpal collapse (a 5.4% ± 2.8% reduction
in the carpometacarpal index, p < 0.05) was observed in the group-1 animals
by six weeks. In contrast, the carpal height was maintained in the group-2 and
3 animals. There was no radiographic evidence of ossification in the group-1
or 2 animals, whereas there was radiographic evidence of ossification in all
six group-3 rabbits killed at the twelve-week time-point. Histologic sections
from the group-3 animals showed filling of the lunate space with islands of
cartilage with interspersed bone ossicles at six weeks. At twelve weeks, there
was abundant bone formation as well as evidence of neovascularization. Osseous
tissue was present in the central portions of the constructs while the
periphery was lined with cartilage. In groups 1 and 2, the lunate space was
filled with poorly organized fibrous tissue.
Conclusions: Cartilaginous implants preformed from autologous
mesenchymal stem cells seeded onto biodegradable scaffold can prevent carpal
collapse. The newly formed osteochondral tissue appears to function as an
adequate biologic lunate spacer for at least twelve weeks in this animal
model.
Clinical Relevance: To our knowledge, this is the first report of
whole-bone reconstruction performed with the use of mesenchymal stem cells.
Biologic constructs that are tissue-engineered from mesenchymal stem cells may
be a new alternative for carpal arthroplasty in patients with clinical
conditions such as osteonecrosis.
The carpus is a complex unit of eight bones arranged into proximal and
distal rows. Stability and motion of the wrist depend on the preservation of
capsuloligamentous structures as well as the contact contours of the
articulating carpal bones. Proper carpal alignment and articulation allow
maintenance of wrist flexion-extension and radial-ulnar deviation. Wrist
instability can be caused by a spectrum of disease entities that involve a
loss of normal ligamentous integrity or by osseous deformities secondary to
trauma or end-stage arthritic changes.
Kienböck disease is a progressive wrist disorder characterized by
osteonecrosis of the lunate that can cause debilitating pain and loss of
function. With advanced disease, carpal collapse is seen with proximal
migration of the capitate and rotatory subluxation of the scaphoid. This leads
to degenerative changes of the intercarpal and radiocarpal joints. The choice
of treatment depends largely on the severity of the disease. However, there is
currently no ideal treatment for advanced lunate collapse. In order to
maintain carpal alignment and prevent carpal instability, a number of
procedures have been described for treatment of advanced stages of
Kienböck disease. These include intercarpal
arthrodesis1-3,
proximal row
carpectomy4-7,
and implantation of a vascularized
graft8,9.
Techniques for direct bone replacement such as use of tendon interposition
grafts10,11
and insertion of prosthetic
implants10,12,13
have demonstrated less than optimal results.
Cell-based tissue-engineering strategies for repair might be a future
alternative for treatment of wrist disorders. Mesenchymal stem cells capable
of multilineage differentiation have been identified in various tissues,
including bone marrow, adipose tissue, and
synovium14-16.
In vitro culture expansion can lead to the growth of billions of mesenchymal
stem cells from as little as 5 to 10 mL of bone marrow
aspirate14. Human
mesenchymal stem cells retain their pluripotentiality and proliferative
ability despite long-term passaging and
cryopreservation14,17.
The bone-marrow harvest process is relatively easy, with minimal donor site
morbidity, and there is potentially a limitless supply of cells. Bone and
cartilage tissue-engineering strategies involving use of mesenchymal stem
cells have been explored in various preclinical animal
models18-21.
Several studies have suggested that repair of full-thickness articular
cartilage defects and subchondral bone may be enhanced by implantation of
mesenchymal stem
cells19,20.
The challenge in treating patients with end-stage arthritis involving the
carpal bones or those with an osseous defect at an articular surface following
trauma is to restore lost bone and the surrounding articular cartilage. We are
not aware of any previous studies demonstrating the possibility of using
mesenchymal stem cells to reconstitute a complete bone with articular
surfaces. The purpose of our study was to determine whether autologous
mesenchymal stem cells can be used to prefabricate a tissue-engineered
cartilage construct that can progress through endochondral ossification to
form a whole bone that will be effective in carpal reconstruction.
Part I: In Vitro Culture of Composite Scaffolds
Cell Culture
Bone-marrow aspirates were obtained from the posterior iliac crest of
skeletally mature New Zealand White rabbits (four to six months old) and then
were processed as previously described to obtain mesenchymal stem
cells22. At 80%
confluence, the cells were then passaged and expanded in medium supplemented
with 10 ng/mL of fibroblast growth factor-2 (FGF-2). Mesenchymal stem cells
treated with FGF-2 in the culture-expansion process have been shown to
proliferate more rapidly and to retain their potential for osteogenic and
chondrogenic
differentiation23-25.
Scaffold Composition
The scaffold was a composite sponge of 70% esterified hyaluronan and 30%
gelatin. The hyaluronan component was obtained from the commercially available
product Laserskin (Fidia Advanced Biopolymers, Abano Terme, Italy). The
gelatin component was hydrolyzed bovine collagen (Sigma, St. Louis, Missouri).
The composite scaffold had pores with two different size ranges of 50 to 150
µm and 250 to 500 µm in
diameter26.
In Vitro Assay
Adherent, passage-one mesenchymal stem cells were trypsinized, washed, and
resuspended in a serum-free defined medium, at a final concentration of
105 cells/µL. The cell suspension was loaded onto
gamma-sterilized composite scaffolds (5 × 4 × 2 mm) and
preincubated for two hours at
37°C22.
Cell-loaded composites were then cultured in a serum-free defined chondrogenic
medium containing transforming growth factor-beta 1 (TGF-ß1) (R and D
Systems, Minneapolis, Minnesota) (10 ng/mL), dexamethasone (10-7
M), ascorbate 2-phosphate (37.5 µg/mL), and ITS + Premix (insulin [6.25
µg/mL], transferrin [6.25 µg/mL], selenious acid [6.25 µg/mL], and
linoleic acid [5.35 µg/mL], with bovine serum albumin [1.25 µg/mL]) (BD
Biosciences, San Jose, California) with medium changes every other
day22,27.
After twenty-one days, replicate constructs were harvested for histologic and
immunohistochemical analysis.
Histologic and Immunohistochemical Analysis
Cell-loaded constructs were fixed in 10% neutral buffered formalin,
embedded in paraffin, and cut into 6-µm sections. Sections were
deparaffinized and rehydrated prior to staining in a 0.03% toluidine-blue
solution for five minutes. Immunohistochemistry was done on replicate
unstained sections. Nonspecific background was blocked with 5% bovine serum
albumin in phosphate-buffered saline solution for thirty minutes. Sections
were then digested in 1 mg/mL of pronase (Roche Diagnostics, Pleasanton,
California) for thirty minutes. Type-II collagen immunohistochemistry was
performed with a monoclonal mouse anti-type-II collagen antibody (II-II6B3,
from the Hybridoma Bank, Iowa City, Iowa) in phosphate-buffered saline
solution containing 1% bovine serum albumin for one hour at room temperature.
Reactivity was detected by incubating with an FITC (fluorescein
isothiocyanate)-linked goat anti-mouse IgG (immunoglobulin G) secondary
antibody (ICN Biomedicals, Costa Mesa, California) for forty-five minutes at
room temperature. Nonspecific reactivity was detected with mouse IgG in place
of primary antibody. Sections were mounted with use of a water-soluble
mounting medium and were examined under light and fluorescent microscopy.
Part II: Lunate Arthroplasty
Lunate Excision
Approval was obtained from the Institutional Animal Care and Use Committee
before the animal procedures were begun. One forelimb from each of
twenty-seven New Zealand White rabbits was shaved, prepared, and draped, and a
tourniquet was applied. A 2-cm dorsal longitudinal skin incision centered over
the lunate was made in line with the third metacarpal. Extensor tendons were
carefully retracted, after which an incision was made through the dorsal fat
pad and joint capsule lying directly over the lunate to identify the
scapholunate and lunatocuneiform joints. The entire lunate was excised. The
animals were divided into three groups
(Table I). In group 1, the
lunate space was left empty. In group 2, a 5 × 4 × 2-mm composite
scaffold without cells was implanted into the lunate space. The size of the
scaffold was based on the approximate size of the lunate in animals in our
preliminary dissection studies. In the group-3 animals, an autologous
mesenchymal stem cell-loaded scaffold construct that had been preincubated in
chondrogenic medium to produce cartilaginous tissue was implanted into the
lunate space. Following placement of the implant, the dorsal joint capsule and
fat pad were reapproximated to secure the construct in the lunate space, after
which the extensor retinaculum and the skin were closed. The wound was then
covered with a soft compressive dressing with no additional immobilization.
The rabbits were allowed to bear weight as tolerated. The group-1 animals were
killed at six weeks, whereas the group-2 and 3 animals were killed at six or
twelve weeks. Tissues were harvested for radiographic and histologic
analysis.
Radiographic Analysis
Standard posteroanterior radiographs of the wrist were made immediately
after surgery and at the time that the animals were killed. The
carpometacarpal index was determined by measuring the ratio between the length
of the third metacarpal and the size of the carpal space (i.e., the distance
between the base of the third metacarpal and the most distal aspect of the
radius) (Fig. 1). Two different
observers used electronic digital calipers to make two sets of measurements,
at least one week apart, with the radiographs arranged in random order. The
mean values were recorded. In order to verify the reproducibility of the
measurements, interobserver reliability was determined by calculating the
Pearson correlation coefficient (R2) between the measurements made
by the two observers. A paired Student t test was used to analyze the change
in the carpometacarpal index between the postoperative radiographs and the
radiographs made when the animals were killed. An unpaired Student t test was
used to compare the carpometacarpal indices among the different groups.
Significance was set at p < 0.05. A power analysis was not performed.
Histologic Analysis
The rabbit wrists were harvested and were embedded in
polymethylmethacrylate. Sections were then cut coronally into 150-µm-thick
slices. Toluidine-blue staining was performed by incubating the sections in
0.03% toluidine-blue solution for five minutes
(Fig. 2). Histologic sections
were reviewed by the authors in a nonblinded fashion.
Part I: In Vitro Culture of Cell-Scaffold Construct
Cell-loaded scaffolds cultured in chondrogenic medium retained their
original size and shape with minimal swelling or distortion of the
three-dimensional architecture. After twenty-one days in vitro, the
cell-seeded scaffold composites exhibited cartilage-like morphology. Evidence
of chondrogenic differentiation throughout the construct was confirmed by the
presence of extensive toluidine-blue metachromasia and type-II-collagen
immunohistochemical staining (Figs. 3-A and
3-B). No cross-reactivity between type-II antibody and the
scaffold was detected.
Part II: Lunate Arthroplasty with Use of Cell-Seeded Scaffold
Construct
Radiographic Analysis
In the group-1 rabbits, in which a lunate excision had been performed
without implantation of a scaffold, carpal collapse occurred by six weeks,
with a mean decrease in the carpometacarpal index of 5.4% ± 2.8% (p
< 0.05) compared with the index on radiographs made immediately after the
surgery (Fig. 4). In contrast,
the lunate space was maintained in group-2 rabbits. In those animals, the
change in the carpometacarpal index between the postoperative and six-week
intervals (1.9% ± 2.3%) was not significant (p = 0.25), with the
numbers studied, and the change between the postoperative and twelve-week
intervals (0.2% ± 1.0%) was also not significant (p = 0.68). Similarly,
the group-3 rabbits, which had undergone lunate excision followed by insertion
of the cell-seeded scaffold construct, were not observed to have significant
carpal collapse, with the numbers studied, at either six weeks (mean change in
the carpometacarpal index, 0.3% ± 0.7%; p = 0.31) or twelve weeks (mean
change, 0.1% ± 2.0%; p = 0.92). There was good interobserver
reliability of the radiograph measurements, with a Pearson product correlation
coefficient (R2) of 0.8662.
No radiographic evidence of bone formation was seen in any of the group-1
or group-2 animals. Evidence of new-bone formation was observed in all six
group-3 animals killed at twelve weeks
(Figs. 5-A and 5-B) but not in
any of the six animals killed at six weeks.
Histologic Analysis
Toluidine blue-stained sections of group-1 rabbit wrists revealed lunate
spaces that were filled with disorganized fibrous tissue
(Figs. 6-A and 6-B). Similarly,
in the group-2 rabbits, the lunate space was replaced with fibrous tissue at
both the six and the twelve-week time-point
(Figs. 6-C and 6-D). No
evidence of bone or cartilage formation was seen in any of the animals. Small
remnants of the original hyaluronan-gelatin-composite scaffold were noted to
be dispersed within the newly formed scar tissue in the group-2 animals.
In the group-3 rabbits, the lunate space was partially filled with repair
tissue, with intensely stained metachromatic matrix, at the six-week
time-point (Figs. 7-A and
7-B). At higher magnification, the cells resembled differentiated
chondrocytes with surrounding proteoglycan deposition in the extracellular
matrix. Evidence of bone formation was observed, with islands of ossicles
within the lunate space. The remaining areas in the defect were filled with
loose connective tissue. At the twelve-week time-point, endochondral
ossification had occurred in the central portions of the lunate space with
extensive replacement of the chondrocytes by woven bone
(Figs. 7-C and 7-D). Compared
with the six-week animals, the twelve-week rabbits had more newly formed
cartilage and osseous tissue within the lunate space. At the host
bone-construct interfaces, the periphery of the newly formed repair tissue
resembled hyaline cartilage. Higher magnification revealed evidence of
neovascularization with the presence of blood vessels within the biologic
construct.
Cell-based tissue-engineering repair strategies represent a new option for
treatment of damaged skeletal tissue. To date, most of these strategies have
been aimed at repairing parts of bone or cartilage. We are not aware of any
previous attempts to reconstitute a complete bone along with its articular
surfaces with use of mesenchymal stem cells. In this study, we attempted to
replace the lunate bone with a tissue-engineered osteochondral construct to
test the clinical utility of such a replacement strategy.
Several authors have reported success after using preformed cartilage-like
constructs to promote healing of bone
defects28-30.
In a rat calvarial model in which bone defects had been filled with periosteal
progenitor cells, Vacanti et al. noted repair tissue that appeared to be
cartilage grossly and histologically during the early
time-points30. This
tissue then progressed to produce bone through an endochondral ossification
cascade. In order to take advantage of this natural embryonic process, we
chose to preincubate our constructs in a chondrogenic medium instead of an
osteogenic medium. Supplementation of our culture medium with osteoinductive
growth factors such as bone morphogenetic protein-2 (BMP-2) or osteogenic
protein-1 (OP-1) would probably have resulted in more of an osseous construct.
The potential advantages of a cartilaginous implant include the maintenance of
articular cartilaginous surfaces for improved joint motion and the possibility
that vascular invasion would occur as part of the endochondral cascade and
result in vascularized repair tissue.
The purpose of this study was to determine whether a cartilage construct
tissue-engineered from autologous mesenchymal stem cells can undergo
endochondral ossification to form a biologic osteochondral substitute that is
effective in carpal bone replacement. We chose to create such a cartilage
anlage for replacement of the lunate. We expected that this cartilage anlage
would be replaced partly by bone through the process of endochondral
ossification. In vitro analysis of the preincubated mesenchymal stem
cell-seeded scaffolds indicated that the constructs implanted into the lunate
space were composed primarily of cartilage-like tissue. At the six-week
time-point, the lunate space was filled with a mixture of cartilaginous and
loose connective tissue. Very little evidence of bone formation was noted.
There was also substantial variability among the rabbits with regard to the
amount and quality of repair tissue that had formed. In some of the six-week
rabbits, large areas of the lunate space resembled fibrous tissue, whereas the
twelve-week rabbits had more bone and cartilage formation within the lunate
space. There was evidence of neovascularization with formation of new blood
vessels as well as replacement of some of the cartilage repair tissue with
immature, woven bone. Longer-term studies are needed to elucidate the eventual
fate of the repair tissue.
Although the central portions of the lunate space were replaced by osseous
tissue, the periphery of the osteochondral repair tissue remained
cartilaginous. The findings of our study suggest that the cells on the
periphery may not proceed down the endochondral cascade; however, longer-term
studies will be required to test this theory appropriately. It has important
clinical implications for the prevention of undesired fusion to the adjacent
joints. Maintenance of the articular cartilage at the host bone-repair tissue
interface would allow the preservation of joint motion.
There are obvious limitations to the use of a small-animal model to study
tissue-engineering. It is not known whether the strategy described here would
be effective in a large-animal model. One of the challenges with larger
constructs would be the difficulty in delivering the growth factor as well as
diffusion of nutrients and metabolic products. More sophisticated
tissue-engineering strategies such as the use of bioreactors and genetic
manipulation of mesenchymal stem cells may enhance repair by allowing improved
tissue formation and greater localized delivery of osteoinductive, angiogenic,
and proliferative growth factors to the defect site. Studies have shown that
BMP-2-producing bone-marrow mesenchymal stem cells produce new bone and are
effective in promoting healing of segmental bone defects and in inducing
spinal
fusion31,32.
Vascular invasion is a vital step in endochondral ossification, and
mesenchymal stem cells transduced with vascular endothelial growth factor
(VEGF) were found to work synergistically with cells transfected with BMP-4 to
enhance bone-healing in critical-sized calvarial
defects33.
Even though the rabbit wrist is a weight-bearing joint, progressive
collapse of the carpus did not occur in the animals treated with the
replacement implant. Allowing the rabbits to bear weight may have enhanced the
formation of osteochondrogenic repair tissue within the lunate space. In fact,
several studies have demonstrated that mechanical loading upregulates
chondrogenesis and osteogenesis in in vitro tissue-engineered
constructs34-38.
In addition to formation of a newly regenerated lunate with cartilage
contours, it appears that the newly formed osteochondral tissue has enough
structural integrity to maintain carpal alignment and prevent carpal collapse
for at least twelve weeks in this animal model. Although the group-2 animals
showed no evidence of new bone or cartilage formation, carpal height was
preserved by the replacement of the scaffold with organized fibrous tissue. It
is possible that the gelatin-hyaluronan construct could function
satisfactorily as a spacer. In the long term, however, we would expect the
fibrous repair tissue in the group-2 animals to be less resistant to
compressive forces when compared with the osteochondral repair tissue seen in
the group-3 animals. Our short-term follow-up study revealed no difference in
the histomorphological characteristics of the fibrous repair tissue or the
degree of carpal height collapse between the six and twelve-week group-2
animals.
Bone-marrow-derived autologous mesenchymal stem cells are readily available
and pose no risk of disease transmission, making them ideal cells for
tissue-engineering. We demonstrated that, with available technology, bone and
cartilage can be created by these cells in a biologically relevant site.
Despite some obvious shortcomings of this animal model, whole-bone replacement
should be explored as a strategy for situations in which a normal shape of the
entire bone is critical. This study is an important first step toward
developing an alternative strategy for treatment of carpal collapse. Although
we could not completely reconstitute an exact lunate, we were able to engineer
an osteochondral construct that may function well as a biologic substitute.
Facilitation of the endochondral process allows the formation of blood vessels
within the neotissue. This provides the possibility of replacing larger bone
defects with use of this process, while avoiding the difficulty of providing
effective vascular conduits for larger in vitro tissue-engineered bone
implants. This will potentially circumvent the need to engineer complex
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