Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study.
ABSTRACT A common approach for tissue regeneration is cell delivery, for example by direct transplantation of stem or progenitor cells. An alternative, by recruitment of endogenous cells, needs experimental evidence. We tested the hypothesis that the articular surface of the synovial joint can regenerate with a biological cue spatially embedded in an anatomically correct bioscaffold.
In this proof of concept study, the surface morphology of a rabbit proximal humeral joint was captured with laser scanning and reconstructed by computer-aided design. We fabricated an anatomically correct bioscaffold using a composite of poly-epsilon-caprolactone and hydroxyapatite. The entire articular surface of unilateral proximal humeral condyles of skeletally mature rabbits was surgically excised and replaced with bioscaffolds spatially infused with transforming growth factor beta3 (TGFbeta3)-adsorbed or TGFbeta3-free collagen hydrogel. Locomotion and weightbearing were assessed 1-2, 3-4, and 5-8 weeks after surgery. At 4 months, regenerated cartilage samples were retrieved from in vivo and assessed for surface fissure, thickness, density, chondrocyte numbers, collagen type II and aggrecan, and mechanical properties.
Ten rabbits received TGFbeta3-infused bioscaffolds, ten received TGFbeta3-free bioscaffolds, and three rabbits underwent humeral-head excision without bioscaffold replacement. All animals in the TGFbeta3-delivery group fully resumed weightbearing and locomotion 3-4 weeks after surgery, more consistently than those in the TGFbeta3-free group. Defect-only rabbits limped at all times. 4 months after surgery, TGFbeta3-infused bioscaffolds were fully covered with hyaline cartilage in the articular surface. TGFbeta3-free bioscaffolds had only isolated cartilage formation, and no cartilage formation occurred in defect-only rabbits. TGFbeta3 delivery yielded uniformly distributed chondrocytes in a matrix with collagen type II and aggrecan and had significantly greater thickness (p=0.044) and density (p<0.0001) than did cartilage formed without TGFbeta3. Compressive and shear properties of TGFbeta3-mediated articular cartilage did not differ from those of native articular cartilage, and were significantly greater than those of cartilage formed without TGFbeta3. Regenerated cartilage was avascular and integrated with regenerated subchondral bone that had well defined blood vessels. TGFbeta3 delivery recruited roughly 130% more cells in the regenerated articular cartilage than did spontaneous cell migration without TGFbeta3.
Our findings suggest that the entire articular surface of the synovial joint can regenerate without cell transplantation. Regeneration of complex tissues is probable by homing of endogenous cells, as exemplified by stratified avascular cartilage and vascularised bone. Whether cell homing acts as an adjunctive or alternative approach of cell delivery for regeneration of tissues with different organisational complexity warrants further investigation.
New York State Stem Cell Science; US National Institutes of Health.
- SourceAvailable from: Catherine Bauge[Show abstract] [Hide abstract]
ABSTRACT: Transforming growth factor beta (TGFb) is a major signalling pathway in joints. This superfamilly is involved in numerous cellular processes in cartilage. Usually, they are considered to favor chondrocyte differentiation and cartilage repair. However, other studies show also deleterious effects of TGFb which may induce hypertrophy. This may be explained at least in part by alteration of TGFb signaling pathways in aging chondrocytes. This review focuses on the functions of TGFb in joints and the regulation of its signaling mediators (receptors, Smads) during aging and osteoarthritis.Aging and Disease. 04/2014; 5(2).
- [Show abstract] [Hide abstract]
ABSTRACT: Controlled release of TGF-β1 from scaffolds is an attractive mechanism to modulate the chondrogenesis of human bone marrow mesenchymal stem cells (hBMSCs) that repopulate articular cartilage defects. Here, we evaluated the ability of porous scaffolds composed of poly(ethylene oxide)-terephtalate and poly(butylene terepthalate) (PEOT/PBT) to release bioactive TGF-β1 for chondrogenesis of hBMSCs in a pellet culture model. Chondroinduction was compared with that promoted by direct addition of the recombinant factor to the culture medium. The data show a controlled release of TGF-β1 from scaffolds for at least 21 days in vitro, with ~ 10% of TGF-β1 released during this period. The delivered TGF-β1 was bioactive, as confirmed by successful chondrogenic differentiation of hBMSCs monitored by morphological, histological, immunohistochemical, biochemical, and real-time RT-PCR analyses. Third, semi-quantitative histological evaluations revealed a similar pattern of chondrogenesis compared with the positive controls. Importantly, TGF-β1-loaded scaffolds allowed for a ~700-fold upregulation of type-II collagen mRNA compared to when pellets were maintained in the presence of the soluble TGF-β1, reflected also in the highest score of immunoreactivity to type-II collagen, not significantly different from the positive controls. Likewise, aggrecan mRNA was ~200-fold upregulated. Interestingly, most (> 94%) of the GAG produced remaining associated with the pellets. Analysis of hypertrophic events showed no significant difference in the average total hypertrophy score compared with the positive controls. These results demonstrate the suitability of controlled TGF-β1 release from biocompatible scaffolds to promote hBMSC chondrogenesis at a physical distance and in the absence of soluble TGF-β1.Journal of Biomedical Materials Research Part A 03/2014; · 2.83 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Purpose: It is well known that implanting a bioactive scaffold into a cartilage defect site can enhance cartilage repair after bone marrow stimulation (BMS). However, most of the current scaffolds are derived from xenogenous tissue and/or artificial polymers. The implantation of these scaffolds adds risks of pathogen transmission, undesirable inflammation and other immunological reactions, and ethical issues in clinical practice. The current study was undertaken to evaluate the effectiveness of implanting autologous bone marrow mesenchymal stem cell-derived extracellular matrix (aBMSC-dECM) scaffolds after BMS for cartilage repair. Methods: Full osteochondral defects were performed on the trochlear groove of both knees in 24 rabbits. One group underwent BMS only in the right knee (the BMS group), and the other group was treated by implantation of the aBMSC-dECM scaffold after BMS in the left knee (the aBMSC-dECM scaffold group). Results: Better repair of cartilage defects was observed in the aBMSC-dECM scaffold group than in the BMS group according to gross observation, histological assessments, immunohistochemistry, and chemical assay. The glycosaminoglycan and DNA content, the distribution of proteoglycan, and the distribution and arrangement of type II and I collagen fibers in the repaired tissue in the aBMSC-dECM scaffold group at 12 weeks after surgery was similar to that surrounding normal hyaline cartilage. Conclusions: Implanting aBMSC-dECM scaffolds can enhance the therapeutic effect of BMS on articular cartilage repair, and this combination treatment is a potential method for successful articular cartilage repair.Tissue Engineering Part A 03/2014; · 4.64 Impact Factor
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X 1
July 29, 2010
Columbia University Medical
Center, Tissue Engineering and
Laboratory, New York, NY, USA
(C H Lee PhD, A Mendelson MS,
E K Moioli PhD, Prof J J Mao PhD);
University of Missouri, College
of Veterinary Medicine and
School of Medicine,
Laboratory, Columbia, MO,
USA (J L Cook DVM); and
Clemson University and
Medical University of South
Carolina, Department of
Bioengineering, Charleston, SC,
USA (H Yao PhD)
Prof Jeremy J Mao, Columbia
University Medical Center,
630 W 168 St – PH7E, New York,
NY 10032, USA
Regeneration of the articular surface of the rabbit synovial
joint by cell homing: a proof of concept study
Chang H Lee, James L Cook, Avital Mendelson, Eduardo K Moioli, Hai Yao, Jeremy J Mao
Background A common approach for tissue regeneration is cell delivery, for example by direct transplantation of stem
or progenitor cells. An alternative, by recruitment of endogenous cells, needs experimental evidence. We tested the
hypothesis that the articular surface of the synovial joint can regenerate with a biological cue spatially embedded in an
anatomically correct bioscaffold.
Methods In this proof of concept study, the surface morphology of a rabbit proximal humeral joint was captured with
laser scanning and reconstructed by computer-aided design. We fabricated an anatomically correct bioscaffold using
a composite of poly-ε-caprolactone and hydroxyapatite. The entire articular surface of unilateral proximal humeral
condyles of skeletally mature rabbits was surgically excised and replaced with bioscaffolds spatially infused with
transforming growth factor β3 (TGFβ3)-adsorbed or TGFβ3-free collagen hydrogel. Locomotion and weightbearing
were assessed 1–2, 3–4, and 5–8 weeks after surgery. At 4 months, regenerated cartilage samples were retrieved from
in vivo and assessed for surface fissure, thickness, density, chondrocyte numbers, collagen type II and aggrecan, and
Findings Ten rabbits received TGFβ3-infused bioscaffolds, ten received TGFβ3-free bioscaffolds, and three rabbits
underwent humeral-head excision without bioscaffold replacement. All animals in the TGFβ3-delivery group fully
resumed weightbearing and locomotion 3–4 weeks after surgery, more consistently than those in the TGFβ3-free
group. Defect-only rabbits limped at all times. 4 months after surgery, TGFβ3-infused bioscaffolds were fully covered
with hyaline cartilage in the articular surface. TGFβ3-free bioscaffolds had only isolated cartilage formation, and no
cartilage formation occurred in defect-only rabbits. TGFβ3 delivery yielded uniformly distributed chondrocytes in a
matrix with collagen type II and aggrecan and had significantly greater thickness (p=0·044) and density (p<0·0001)
than did cartilage formed without TGFβ3. Compressive and shear properties of TGFβ3-mediated articular cartilage
did not differ from those of native articular cartilage, and were significantly greater than those of cartilage formed
without TGFβ3. Regenerated cartilage was avascular and integrated with regenerated subchondral bone that had well
defined blood vessels. TGFβ3 delivery recruited roughly 130% more cells in the regenerated articular cartilage than
did spontaneous cell migration without TGFβ3.
Interpretation Our findings suggest that the entire articular surface of the synovial joint can regenerate without cell
transplantation. Regeneration of complex tissues is probable by homing of endogenous cells, as exemplified by
stratified avascular cartilage and vascularised bone. Whether cell homing acts as an adjunctive or alternative approach
of cell delivery for regeneration of tissues with different organisational complexity warrants further investigation.
Funding New York State Stem Cell Science; US National Institutes of Health.
Tissue or organ defect is the final outcome of all incurable
diseases. An organ consists of dissimilar tissues, and
every tissue is formed of one or more cell types. For
decades, cell-based approaches have been used to replace
diseased cells and to heal tissue defects.1–5 Cell delivery
has, however, encountered crucial barriers in therapeutic
translation, including immune rejection, pathogen
transmission, potential tumorigenesis, issues with
packaging, storage, and shipping, and difficulties in
clinical adoption and regulatory approval.6–8 Tissue
regeneration by recruitment of the host’s endogenous
cells, including stem or progenitor cells, is an emerging
idea. However, tissue regeneration by cell homing,
especially without cell transplantation, remains a
provocative approach in need of experimental testing.9–12
The synovial joint consists of multiple tissues including
articular cartilage, subchondral bone, haemtopoietic
marrow, and synovium. Osteoarthritis manifests as
structural breakdown of cartilage and bone, and is a
leading chronic disability worldwide, affecting about
80 million individuals in the USA alone.13,14 At present,
arthritic joints are replaced by total joint arthroplasty using
metallic and synthetic materials. Existing joint prostheses
fail mainly because of aseptic loosening or infection
induced by wear debris.13–15 Given an average lifespan of
10–15 years, metal prosthesis is problematic in the
substantial and increasing population of arthritis patients
who are aged 65 years or younger.13–15 Furthermore, metal
prostheses do not remodel with host bone, leading to
aseptic loosening, an issue that can only be solved by
biological regeneration. Similar to regeneration of other
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X
tissues, cartilage regeneration is replete with examples of
cell delivery.16–19 The predominant model of cartilage
regeneration is focal lesions.20,21 However, focal lesions
deteriorate, with or without existing interventions, into
severe arthritis that ultimately warrants total joint
arthroplasty.15,22,23 Commercial approaches for the treatment
of focal lesions in patients are only partly successful and
are associated with persistent drawbacks such as
suboptimum integration, loss of chondrocyte phenotype,
and guarded functional outcome.22–24 We tested the
Figure 1: Surgical replacement of synovial joint
Surface morphology of a rabbit joint was reconstructed (A) to design an anatomically correct bioscaffold (B) with an intramedullary stem. A 200-µm thick shell was
designed, along with internal microchannels opening to the synovium cavity (C) and bone marrow (D). PCL-HA was used to fabricate bioscaffolds following
computer-aided design (E). The humeral head was excised at its metaphysis junction (F), and an orthopaedic drill used to create an intramedullary tunnel for stem
fixation (G). The bioscaffold (H) was implanted by press-fitting (I). In defect-only rabbits, haematoxylin and eosin staining 4 months after surgery (J) showed that
little bone had regenerated in the defect; the synovial joint cavity (sc) was visible with fibrous tissue (f) covering bone and marrow (m). Safranin O staining (K)
showed scarce chondrocyte-like cells (shown by arrows) in the defect area in the synovial cavity. PCL-HA=poly-ε-caprolactone hydroxyapatite.
400 µm400 µm
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X 3
hypothesis that the entire articular surface of the synovial
joint can regenerate with a bioactive cue spatially embedded
in an anatomically correct bioscaffold.
Design and fabrication of the bioscaffold
Surface morphology of the cadaver humeral head of the
forelimb joint from a 6-month-old rabbit (matching those
used for in-vivo experiments) was captured with multislice
laser scanning at 12·7 µm resolution (Berding, Loveland,
OH, USA; figure 1A), and reconstructed in three dimensions
by computer-aided design (figure 1E). An anatomically
correct bioscaffold measuring 12·42×10·11×16·88 mm
(length×width×height) was designed to replace the condylar
head, with an intramedullary stem for surgical fixation
(figure 1B). This overall dimension is far greater than the
capacity for diffusion exchange of regenerating tissues,
which is about 200 µm.25 Accordingly, we designed three-
dimensional interconnecting microchannels (200–400 µm
diameter) as conduits to promote tissue regeneration
(figure 1C, D). We fabricated the bioscaffold using three-
hydroxyapatite (HA) powder (Sigma, St Louis, MO, USA)
into 80 wt% poly-ε-caprolactone (PCL; molecular weight
roughly 65 000, Sigma) slurry at 120°C (Bioplotter,
EnvisionTec, Gladbeck, Germany). We selected PCL-HA in
accordance with our previous finding of cell adhesion and
osteochondral histogenesis using the same materials.26
Figure 1C shows the superior portion of the bioscaffold,
with the top 500 µm designed for regeneration of cartilage,
similar to the thickness of the native rabbit humeral articular
cartilage. Figure 1D shows the inferior portion, which was
designed for regeneration of bone, with the intramedullary
stem omitted for demonstration. Interlaid strands and
interconnecting microchannels had diameters of 400 µm in
the cartilage portion (figure 1C), and 200 µm in the bone
portion (figure 1D).
bioprinting; 20 wt%
Transforming growth factor β3 (TGFβ3) at a dose of
10 ng/mL (Cell Biosciences, Palo Alto, CA, USA) was
adsorbed in 5 mg/mL neutralised collagen type I
(Cultrex, R&D, Minneapolis, MN, USA). We selected
this dose of TGFβ3 to stimulate chondrogenic
differentiation.26,27 TGFβ3-adsorbed or TGFβ3-free
collagen solution was infused into the microchannels of
the bioscaffold to a depth of roughly 500 µm, and cross-
linked for 1 h at 37°C without additional cross-linker.
Our rationale for 400-µm diameter microchannels in
the cartilage portion of the scaffold was to provide space
for matrix synthesis and anchorage for TGFβ3-loaded
or TGFβ3-free collagen gels.
Surgical joint replacement
We received Institutional Animal Care and Use Committee
approval to use skeletally mature New Zealand white
rabbits (aged 6 months, weighing 3·5–4·0 kg; Harlan,
Indianapolis, IN, USA). Skeletal maturity was defined by
age (5–6 months) and bodyweight (3·5–4·0 kg), following
the established criteria for cartilage regeneration.15 The
rabbits were anaesthetised with ketamine (35 mg/mL) and
xylazine (5 mg/mL), and maintained with 1–5% isoflurane.
With a craniolateral approach, the acromial head of the
deltoid muscle was tenotomised at its origin and retracted
distally. The infraspinatus muscle was tenotomised at its
insertion and retracted caudally. The lateral joint capsule
was incised from cranial to caudal to expose the humeral
head by internal rotation and complete lateral luxation. An
osteotome and mallet were used to excise the humeral
head at its metaphysis junction, while preserving the
greater and lesser tubercles and all soft tissue attachments
(figure 1F) to simulate unipolar joint arthroplasty. A
3·2 mm drill bit on a hand chuck created an intramedullary
tunnel for stem insertion (figure 1G). After humeral-head
excision, the anatomically correct bioscaffold (figure 1H)
was implanted by press-fitting (figure 1I). The joint capsule
was closed with a mattress suture, followed by reattachment
of the infraspinatus and deltoid tendons. The subcutis was
apposed with 4-0 polydioxanone suture, followed by skin
closure. Locomotion and weightbearing were assessed at
1–2, 3–4, 5–8 weeks postsurgery.
Histology and histomorphometric analysis
4 months after implantation, articular cartilage samples
were stained with India ink and imaged with a high-
resolution camera to reveal surface fissures and defects.
Harvested tissue and scaffold samples were embedded in
poly(methyl methacrylate) and cut into 5-µm sections
(HSRL, Jackson, VA, USA). Using a random number
table, we selected sections to be separately stained with
safranin O, von Kossa, and haematoxylin and eosin.
Cartilage thickness was measured as the linear distance
from articular surface to subchondral bone at ten equally
spaced locations across the entire cartilage. The areal
matrix density was measured as the intensity of safranin
O staining across the entire cartilage with the same
microscope contrast and brightness. We counted the
numbers of cells in regenerated cartilage on ten randomly
selected microscopic sections per sample in each of the
groups. The number and diameter of blood vessels with
erythrocytes and lined by endothelial cells in regenerated
subchondral bone were quantified by image analysis. All
measurements were done separately by two calibrated
and masked examiners. The glenoid fossae, which are
the opposing articular surfaces, were harvested and fixed
in 4% formaldehyde. After paraffin embedding, we cut
5-μm serial sections in the medial, central, and lateral
planes (webappendix p 4).
Immunofluorescence of collagen type II and aggrecan
4 months after in-vivo implantation, we immunoblotted
collagen type II and aggrecan in the cartilage matrix.
Tissue samples were washed with 0·1% Triton X and
incubated with monoclonal antibodies for collagen type
See Online for webappendix
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X
II (1:100; CP18, Calbiochem, Gibbstown, NJ, USA) or
aggrecan (1:100; ab3773, Abcam, Cambridge, MA, USA)
for 1·5 h at room temperature. Before application of
aggrecan antibody, samples
chondroitinase ABC, keratanase, and keratanase II for
1 h. After 1-h incubation with secondary antibodies of
Alexa Fluor 680 (Invitrogen, Carlsbad, CA, USA) and
IRDye 800CW (LI-COR, Lincoln, NE, USA), we
semiquantified samples using infrared imaging with
700-nm excitation and 800-nm emission wavelengths
(Odyssey, LI-COR). The integrated intensity of
fluorescence normalised to each immunoreactive area
was calculated as relative immunoreactivity.
were treated with
Mechanical properties of regenerated cartilage
We did compressive and shear tests using an ElectroForce
BioDynamic Tester (Bose, Eden Prairie, MN, USA).
Cylindrical osteochondral plugs (5·05 [SD 0·51]×5·21
[0·31] mm2) were punched from native, TGFβ3-
regenerated, and TGFβ3-free cartilage samples 4 months
after in-vivo implantation. Compressive testing was done
under 10% cyclic strain at 2 Hz, and shear testing with
3% sinusoidal strain under 10% compressive strain at
2 Hz.28 We characterised dynamic compressive properties
as complex compressive modulus E*, storage modulus
E’, and loss modulus E”, and dynamic shear properties as
the complex shear modulus G*, shear storage modulus
G’, and shear loss modulus G”.
All statistical analyses were done with SPSS (version 16)
and PASS 2005. Power analyses were performed with a
significance level of 0·05, effect size of 1·50, and power
of 0·8. Effect size and power calculations were based on
our pilot experiments and previous work.26 For normal
distribution, quantitative data for control and treated
groups were subjected to one-way ANOVA and post-hoc
least significant difference tests. For skewed data, we
used non-parametric Kruskal-Wallis tests.
Role of the funding source
The funding source was not involved in the design, data
collection, data analysis, data interpretation, or writing of
the report. The corresponding author had full access to
all data in the study and had final responsibility for the
decision to submit for publication.
Ten rabbits received anatomically correct bioscaffolds
infused with TGFβ3-adsorbed collagen gel, and ten
received bioscaffolds infused with TGFβ3-free gel. Three
animals underwent condyle excision without bioscaffold
replacement. Within the first 1–2 weeks after joint
replacement, the representative TGFβ3-infused rabbit
limped with little use of the operated right forelimb
(webvideo 1). By 3–4 weeks postsurgery, the same rabbit
underwent locomotion and weightbearing with all limbs
Figure 2: Articular cartilage regeneration
India ink staining of (A) unimplanted bioscaffold, (B) TGFβ3-free and (C) TGFβ3-infused bioscaffolds after 4 months’ implantation, and (D) native cartilage. (E)
Number of chondrocytes present in TGFβ3-infused and TGFβ3-free regenerated articular cartilage samples (n=8 per group). Safranin O staining of TGFβ3-free (F,I) and
TGFβ3-infused (G,J) articular cartilage. Matrix density (H) and cartilage thickness (K) of TGFβ3-infused and TGFβ3-free samples (n=8 per group for both comparisons).
TGFβ3=transforming growth factor β3. PCL-HA=poly-ε-caprolactone hydroxyapatite.
Number of cells per mm2
Cartilage thickness (μm)
200 µm 200 µm
See Online for webvideos
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X 5
including the operated one (webvideo 2). By 5–8 weeks
postsurgery, the same rabbit moved (webvideo 3) almost
as unoperated, age-matched rabbits. These webvideos
are representative of many video-recorded animals.
4 months after humeral-head excision without
bioscaffold implantation, there was little regeneration in
the defect, showing synovial cavity with residual scarring
fibrous tissue covering immature, woven bone and
marrow (figure 1J). Safranin O staining revealed isolated
chondrocyte-like cells in the defect’s fibrous layer
(figure 1K), but little cartilage formation. Functionally,
defect-only rabbits with condyle excision limped at all
times, suggesting that condylar-head ablation is a critical-
size defect for both cartilage and bone. By contrast,
cartilage and bone regeneration occurred in bioengineered
synovial joint replacements. In reference to an
Figure 3: TGFβ3 delivery and quality of articular cartilage
Immunostaining for Col-II and AGC in unimplanted bioscaffold (A–D) and native (E–H), TGFβ3-free (I–L), and TGFβ3-infused (M–P) articular cartilage samples.
Immunoreactivity for Col-II (Q) and AGC (R) in native, TGFβ3-free, and TGFβ3-infused samples (n=10 per group). Col-II=collagen type II. AGC=aggrecan.
TGFβ3=transforming growth factor β3.
Col–II Col–IIAGC AGC
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X
unimplanted bioscaffold sample (figure 2A), the TGFβ3-
infused bioscaffold had full tissue coverage (figure 2C),
whereas the TGFβ3-free bioscaffold had only isolated
tissue formation (figure 2B). The native articular surface
is shown for comparison (figure 2D).
India ink, a dye that reveals surface fissures and defects,
marked the border of microchannel openings of
the unimplanted bioscaffold (figure 2A), but did not stain
either TGFβ3-regenerated cartilage (figure 2C) or native
articular cartilage (figure 2D). By contrast, India ink
stained the border of isolated cartilage tissue of the
TGFβ3-free sample (figure 2B). TGFβ3 delivery recruited
roughly 130% more cells in the regenerated articular
cartilage than did spontaneous cell migration without
TGFβ3 (figure 2E; n=8; p<0·0001), suggesting that
TGFβ3 is a chemotactic cue for cell homing.
Microscopically, chondrocytes in the TGFβ3-free sample
clustered and synthesised matrix with positive but
moderate safranin O staining (figure 2F, I).By contrast,
TGFβ3 delivery yielded evenly distributed chondrocytes
and intense safranin O staining for both pericellular and
interterritorial matrices (figure 2G, J).
Regenerated articular cartilage in the TGFβ3-infused
group extended above the bioscaffold’s superior surface
Figure 4: Vascularisation of regenerated subchondral bone
(A) Radiolucency (shown by arrow) was present in the joint cavity on excision of the condylar head. By 8 weeks (B) and 16 weeks (C) after surgery, a convex radio-
opaque structure in the shape of articular condyle was present. (D) After 16 weeks’ implantation, regenerated cartilage (c) extended to subchondral bone (b), which
consisted of trabecular structures (E). (F, G) Von Kossa staining indicated mineral deposition that extended from the cartilage region (c, blue area) longitudinally in
microchannels (m). (H) Bone trabeculae were populated by columnar osteoblast-like cells (which are shown by arrows). (I) Regenerated bone integrated to native
humeral bone (arbitrary boundary shown by dashed blue line). (J) Multiple blood vessels (example shown by arrow) were present in regenerated bone. High
magnification (K) showed the presence of erythrocytes within the lumen of an endothelium-lined blood vessel (arrow). PCL-HA=poly-ε-caprolactone hydroxyapatite.
Replacement8 weeks 16 weeks
500 µm 200 µm50 µm
200 µm 500 µm200 µm
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X 7
(figure 2C, G), suggesting that cartilage, instead of the
bioscaffold, was articulating with the opposing native
cartilage (glenoid) surface. Quantitatively, samples from
the TGFβ3-infused group had significantly greater matrix
density (figure 2H; p<0·0001) and articular cartilage
thickness (figure 2K; p=0·044) than did samples from the
TGFβ3-free group (n=8 per group for both comparisons).
In either regenerated or native articular cartilage, fast
green counterstaining was absent, suggesting paucity of
fibrous tissue, by comparison with the presence of
fibrous tissue or fibrocartilage in defect-only samples
(figure 1J, K). Regenerated articular cartilage was derived
entirely from host endogenous cells.
Immunostaining showed no autofluorescence of
unimplanted bioscaffold (figure 3A–D). Collagen type II
and aggrecan, two major macromolecules of articular
cartilage, were present in the native cartilage sample
(figure 3E–H). By contrast with uneven and isolated areas
of collagen type II and aggrecan in TGFβ3-free samples
(figure 3I–L), TGFβ3-regenerated samples had consistent
and continuous distribution of both collagen type II and
aggrecan in the articular surface and sagittal plane
(figure 3M–P). Semiquantitatively, TGFβ3 delivery
yielded greater collagen type II and aggrecan
immunoreactivity than was seen either in the TGFβ3-
free or native group (figure 3Q, R; n=10 per group),
probably because of active remodelling. The glenoid
fossae showed qualitatively little sign of cartilage injury
or osteoarthritis in photographs and microscopic images
in the medial, central, and lateral planes of TGFβ3-
delivered or TGFβ3-free samples, by comparison with
the native glenoid fossa (webappendix p 4).
A radiolucent region was present in the joint cavity
after excision of the articular surface and subsequent
implantation of the radiolucent bioscaffold (figure 4A). A
convex, radio-opaque structure was present in the joint
cavity of the same rabbit 8 weeks (figure 4B) and 16 weeks
(figure 4C) after surgery, suggesting the formation of a
mineralised condyle-like structure. The regenerated
articular cartilage extended to subchondral bone
(figure 4D, E). Von Kossa staining showed mineral
deposition within the bioscaffold’s interconnecting
microchannels (figure 4F), extending from articular
cartilage inferiorly along the microchannel wall (figure 4F,
G). Trabecular structures were populated by columnar
osteoblast-like cells (figure 4H). The regenerated
subchondral bone integrated to native bone (figure 4I).
Vasculature was present in regenerated bone
(figure 4J, K), but absent in regenerated articular
cartilage. TGFβ3 delivery did not increase either blood
vessel number (9·2 [SD 3·4] per mm) or vessel diameter
(72·9 [46·7] μm) compared with the TGFβ3-free group
(10·4 [4·5] per mm, 67·1 [28·4] μm; n=8; p=0·284 for
vessel number; p=0·158 for vessel diameter), leading to
a speculation that angiogenesis and osteogenesis can be
further improved. Importantly, all blood vessels were
host-derived. Whole bone and scaffold µCT images
showed thorough host tissue ingrowth in microchannels
in both TGFβ3-free and TGFβ3-infused samples
(webappendix p 5).
4 months after implantation, |E*|, E’, and E” of TGFβ3-
infused articular cartilage samples did not differ from
those of native articular cartilage, but were significantly
higher than those of TGFβ3-free samples (figure 5; n=5
for all groups). Similarly, |G*|, G’ and G” were significantly
higher in TGFβ3-regenerated samples than in TGFβ3-
free samples. |G*| and G’ did not differ between native
and TGFβ3-regenerated cartilage samples, but G” was
significantly higher in native cartilage than in TGFβ3-
regenerated and TGFβ3-free samples. In general, the
dynamic viscoelastic moduli of the TGFβ3-infused
articular cartilage were similar to those of the native
articular cartilage samples.28
Our results show that the entire articular surface of a
synovial joint can be regenerated by homing of
endogenous cells. Webvideos of locomotion and
weightbearing of joint-replaced
substantiated by regeneration of avascular articular
cartilage and vascularised subchondral bone, two
dissimilar tissues that function in unison. We used a
rabbit model, despite the opinion that focal lesions of
rabbit cartilage have spontaneous healing capacity.29
However, spontaneous healing in animals the size of
rabbits or smaller, if present, seems to apply only to
focal defects. Notably, the present model of total condyle
ablation did not spontaneously regenerate and remained
a critical-size defect up to 4 months after condyle
excision. With TGFβ3 delivery, however, cartilage
regenerated over the bioscaffold’s superior surface, and
allowed functional recovery of weightbearing and
locomotion. Regenerated cartilage is probably hyaline,
in view of the presence of collagen type II and aggrecan
| E* |E’ E’’| G* |G’ G’’
Figure 5: Mechanical properties of regenerated cartilage
E*=complex compressive modulus. E’=storage modulus. E”=loss modulus. G*=complex shear modulus. G’=shear
storage modulus. G”=shear loss modulus. TGFβ3=transforming growth factor β3.
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X
and the absence of fast green staining; by contrast,
temporomandibular joint show strong fast green
counterstaining in the superior portion of fibrocartilage.30
The probability of hyaline cartilage regeneration is
further substantiated by the similar mechanical
properties of TGFβ3-regenerated cartilage to those of
native articular (hyaline) cartilage. In general, the
compressive modulus of hyaline cartilage is about four
to eight times greater than that of fibrocartilage.31
Cell homing underlies the regeneration of articular
cartilage in vivo. The process of cell homing has been
used to describe the extravasation of circulating cells.9,10
Here, we broadly define cell homing as the recruitment
of endogenous cells to an anatomic compartment.
Despite TGFβ3’s broad effects on cell adhesion and
differentiation, it is rarely regarded as a cell homing
molecule.30,32,33 We have shown that TGFβ3 infusion
recruits about 130% more cells than in the absence of
TGFβ3. Formation of cartilage in the absence of TGFβ3
is likely due to spontaneous cell migration in the joint
environment, similar to sparse chondrocytes in defect-
only samples; however, cartilage formed in this way has
low concentrations of aggrecan and collagen type II, and
poor mechanical properties. An approach similar to ours,
without growth factor delivery, yielded fibrous tissue or
fibrocartilage scarring and poor functional recovery,34
providing partial confirmation of our TGFβ3-free group
and again underscoring the potency of bioactive cues in
cartilage regeneration. The porous surface at the proximal
end of our bioscaffold in the synovial cavity provided
access for synovium stem or progenitor cells, which are a
probable source of articular chondrocytes,35,36 just as the
porous distal end provided access for bone marrow stem
or progenitor cells.
We speculate that some of the endogenous cells are
derived from stem or progenitor cells of synovium,
bone marrow, adipose (fat pad adjacent to synovial
membrane and bone marrow), and perhaps vasculature.
Although subsequent studies are needed to elucidate
the precise sources of endogenous cells that home to
regenerate articular cartilage and subchondral bone,
we have made the following initial attempts
(webappendix p 6). Cells were separately isolated from
regenerated articular cartilage and subchondral bone.
Cells from regenerated cartilage were spherical, but
assumed spindle shape after 7-day culture, whereas
cells from regenerated bone maintained their initial
spindle shape. Activin A, a marker preferentially
expressed by bone marrow cells,37 showed little
expression in cells from regenerated cartilage, but was
robustly expressed in cells from regenerated bone
(webappendix p 6). Furthermore TGFβ3 recruited a
remarkable number of mesenchymal stem or progenitor
cells and synovium stem or progenitor cells in vitro by
comparison with spontaneous
(webappendix p 7). We suspect that cells from several
such as the
sources were recruited by TGFβ3 in the microchannels
of the bioscaffold and interacted in regeneration.
Delineation of endogenous cell sources will contribute
to strategies that recruit specific cell populations for
regeneration of complex tissues.
We have unravelled several crucial roadblocks in
regenerative medicine including
angiogenesis, scale up, and tissue biophysical
properties. Viscoelastic properties of TGFβ3-mediated
articular cartilage are not only in the same range as
those of native articular cartilage, but are also superior
to those of TGFβ3-free samples, suggesting that
regenerated articular cartilage has sufficient mechanical
stiffness. This finding is confirmed by weightbearing
and locomotion (webvideos 1–3), as well as microscopic
data for articular cartilage
biocompatible PCL-HA used to create the bioscaffold
possesses sufficient mechanical stiffness for initial
weightbearing. PCL can be biodegraded by hydrolysis
of its ester linkages, and can be further modified, if
warranted, with ring-opening polymerisation and
The regeneration of the entire articular surface of a
synovial joint has succeeded probably because of
modularisation of a large tissue scaffold with repeating
and interconnecting microchannels serving as conduits
for cell homing, diffusion,
angiogenesis. Irrespective of scaffold size, each module
contains microchannel or microstrand units in the
range of 200–400 µm, which seems to be a crucial
threshold for survival of regenerating tissues.25 The
present translational strategy provides a feasible
approach for regeneration of dissimilar tissues such as
avascular cartilage and vascularised bone, which
constitute the entire articular surface of a synovial joint.
An important extension of our work is replacement of
arthritic synovial joints in preclinical models and in
arthritis patients who need total joint arthroplasty. Since
cartilage, a tissue that is recalcitrant to regeneration, is
regenerated by endogenous cells that are recruited from
the host, there is reason to believe that other tissues
could also regenerate without cell transplantation.
These findings suggest a regenerative approach for
cartilage and synovial joint defects that can potentially
be translated into patients with arthritis, trauma, or
osteonecrosis. Cell homing, instead of cell delivery,
might accelerate therapeutic translation.
CHL was responsible for the primary technical undertaking and
technical design, did the experiments, and drafted the report. JLC
participated in the overall design, especially the surgical design and
animal model, and did all animal surgeries. AM did the in-vitro cell
homing experiment. EKM contributed to the technical design of the
biomaterials and participated in animal surgery. HY did µCT analysis.
JJM conceived and designed the experiments, and oversaw the collection
of results, data interpretation and finalised the report.
Conflicts of interest
We declare that we have no conflicts of interest.
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60668-X 9
This work was supported by a research grant from the New York State
Stem Cell Science and US National Institutes of Health grant
R01EB002332. We thank ICM staff at Columbia University Medical
Center for veterinary assistance, and members of J J M laboratory for
administrative and additional technical support. Synovium cells were a
generous gift from Elena Jones of University of Leeds, UK.
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www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60931-2 1
July 29, 2010
In-vivo tissue engineering of biological joint replacements
In The Lancet today, Chang Lee and colleagues1
report the successful regeneration of entire humeral
condyles in rabbits after radical resection of the
original structures. Regeneration was achieved by
implantation of customised anatomically shaped
bioscaffolds that were infused with collagen gel
containing transforming growth factor β3 to stimulate
chondrogenic differentiation of cells. No preprepared
stem cells or other cells were added to the scaffold
before implantation. Instead, the investigators relied
on endogenous cell homing, local tissue response,
and functional stimulation to recreate the entire
articulating cartilaginous joint surfaces together with
subchondral bone. Impressively, only 3–5 weeks after
surgery, the rabbits were able to move almost as well
as animals that had not undergone the procedure.
However, this study was in rabbits, not in human
beings. How then are the results relevant to clinicians?
2010 is the closing year of the Bone and Joint Decade,
as designated by the former UN Secretary-General,
Kofi Annan, in November, 1999.2 The decade was
named to draw attention to a worldwide epidemic of
musculoskeletal and joint disease, affecting hundreds
of millions of people.3 This figure was projected to
increase sharply because of the predicted doubling of
the number of people older than 50 years by 2020.4
Osteoarthritis is the leading disease of the joints—it
affects one in eight Americans and almost half the
world’s population older than 65 years.4 Although many
patients with arthritis can be managed conservatively,
surgery and in particular joint-replacement surgery
is often necessary for arthritic joint diseases and
can be a life-changing cure. Unfortunately, today’s
joint replacements are still made of solid titanium or
stainless steel and therefore have a finite lifespan. In
addition to infection of the implant, aseptic loosening
is a particular concern in young active patients. Rates of
clinical failure of 13–20% have been reported for total
hip replacements after 10 years, leading to revision
surgery in around 5–7% of patients.5,6 Revision surgery
is often associated with inferior results, because
compromised local tissue conditions and loss of bone
adversely affect stability.5,6
We have been awaiting advances in joint-replacement
research with the hope that we might one day grow
individually customised biological joint replacements
for patients in the laboratory. There is, of course, a
substantial way to go before such a dream is to be
realised, but there is room for hope. Today’s study
could be a substantial step towards that goal. Lee and
co-workers’ study is a renaissance of use of the host as
a bioreactor and recruitment of the host’s endogenous
cells, including stem or progenitor cells, for tissue
regeneration. They have bucked the current trend
for use of ex-vivo cultivated stem-cell preparations
for tissue engineering. Much hype has surrounded
stem-cell-based tissue engineering; from discussion
in the popular media, it would seem that we are only
just around the corner from amazing solutions to the
shortage of organs available for transplant or growing
new joints. It might seem that all that is needed is to
put the right recipe of stem cells into the petri dish. This
approach might work in theory for simple homogeneous
tissue and in small proportions, but without a vascular
supply, any tissue grown ex vivo requires life support by
complex bioreactors. Experience with this approach to
date has been limited to production of functional tissue
not really greater in size than a sugar cube.
Can the technique described by Lee and colleagues be
directly translated to human beings? Theoretically, yes,
but there are some potential drawbacks. Replacement
of the joint condyle with an anatomically designed
bioscaffold by this technique sounds surgically feasible.
Figure: Use of man as a living bioreactor for the growth of biological joint replacements8
www.thelancet.com Published online July 29, 2010 DOI:10.1016/S0140-6736(10)60931-2
replacement in the latissimus dorsi of a man. The
replacement was subsequently successfully transplanted
to repair a defect after surgery for a tumour in the floor
of the mouth.8,9 In our work, the patient was used as the
bioreactor or endocultivator to grow the replacement
part, proving that endocultivation was feasible for
large bone replacements. The cultivation of bone and
cartilage together with this technique is a challenge
that the multinational tissue engineering network,
MyJoint, funded by the European Union, is working to
overcome.10 This study by Lee and colleagues offers new
insights into in-vivo tissue engineering, especially in
bioscaffold design and endogenous cell homing.
Ultimately, the optimum way to grow a biological joint
replacement remains a controversy at this, the end of
the Bone and Joint Decade. Although we are yet to see a
biological joint replacement in man, Lee and colleagues
have offered a promising insight into what might be on
Patrick H Warnke
Faculty of Health Sciences and Medicine, Bond University,
Gold Coast, QLD 4229, Australia
I am the head and coordinator of the multinational tissue engineering
network “MyJoint”,10 which is funded by the European Union. Several
universities and biomedical institutes are working together to modify a
recently established technique8 to use patients as their own bioreactors to
grow biological joint replacements.
1 Lee CH, Cook JL, Mendelson A, Moioli EK, Yao H, Mao JJ. Regeneration of
the articular surface of the rabbit synovial joint by cell homing: a proof of
concept study. Lancet 2010; published online July 29. DOI:10.1016/S0140-
Brooks PM, Hart JA. The bone and joint decade: 2000–2010.
Med J Aust 2000; 172: 307–08.
Brooks PM. The burden of musculoskeletal disease—a global perspective.
Clin Rheumatol 2006; 25: 778–81.
Weinstein SL. 2000–2010: The bone and joint decade. J Bone Joint Surg Am
2000; 82: 1–3.
Söderman P, Malchau H, Herberts P, Zügner R, Regnér H, Garellick G.
Outcome after total hip arthroplasty: part II. Disease-specific follow-up
and the Swedish National Total Hip Arthroplasty Register.
Acta Orthop Scand 2001; 72: 113–19.
Kärrholm J, Garellick G, Rogmark C, Herberts P. The Swedish Hip
Arthroplasty Register: annual report 2007. http://www.jru.orthop.gu.se
(accessed June 24, 2010).
Cullinane DM, Salisbury KT, Alkhiary Y, Eisenberg S, Gerstenfeld L,
Einhorn TA. Effects of the local mechanical environment on vertebrate
tissue differentiation during repair: does repair recapitulate development?
J Exp Biol 2003; 206: 2459–71.
Warnke PH, Springer ING, Wiltfang J, et al. Growth and transplantation of a
custom vascularised bone graft in a man. Lancet 2004; 364: 766–70.
Warnke PH, Wiltfang J, Springer I, et al. Man as living bioreactor: fate of
an exogenously prepared customized tissue-engineered mandible.
Biomaterials 2006; 27: 3163–67.
10 MyJoint—The Biologic Joint Replacement Project. http://www.myjoint.org
(accessed June 17, 2010).
However, the regenerative capacity of some patients,
especially elderly patients with comorbidities such as
diabetes mellitus, would be expected to be impaired.
If the bioscaffold is not weightbearing during the
generative phase (which seems impractical), patients
would be bedbound for a substantial time. One could
reasonably expect that several weeks or months might
be needed to allow for sufficient tissue ingrowth
for functional loading. The formation of a smooth
articulating cartilage surface would require mechanical
stimulation and loading.7 This process in turn would
require the patient to undertake a substantial physio-
therapy regimen. For most patients, a standard metal
joint replacement is likely to offer a faster and less
demanding option than the bioscaffold, with fewer
risks associated with immobility. Application of Lee and
colleagues’ or a similar technique might, however, offer
important advantages to young patients who need
Of course, there are hurdles to overcome before any
serious discussion about application of this technique
to human beings. Existing technology could potentially
help to overcome these problems. One way to reduce
regeneration time would be to commence cultivation
of the tissue ex vivo in bioreactors and then implant a
premature biological joint replacement. This preformed
and immature joint replacement could provide limited
functional loading, with further tissue stimulation
provided by the recipient. However, as discussed, the
amount of tissue generated in an artificial bioreactor is
unlikely to be sufficient for this purpose. Additionally,
bone and cartilage would require different competing
environmental conditions during cultivation in vitro.
Growing of chondrocytes and osteocytes together in
one culture is therefore challenging and not currently
technically feasible as regards potential use in a
Another promising approach would be to commence
the entire cultivation of the joint replacement inside
the patient, but to change the site of tissue growth
(figure). Tissue at the size of a joint could be grown
inside a muscle first and subsequently transplanted to
replace the original joint. The patient could continue
to use the compromised joint, while simultaneously
growing the new one and taking advantage of in-
vivo cultivation. In 2004, we described the cultivation
of a vascularised individually customised mandible