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Chitosan-Glycerol Phosphate/Blood Implants Improve Hyaline Cartilage
Repair in Ovine Microfracture Defects
Caroline D. Hoemann, Mark Hurtig, Evgeny Rossomacha, Jun Sun, Anik Chevrier, Matthew S. Shive and Michael
D. Buschmann
J. Bone Joint Surg. Am. 87:2671-2686, 2005. doi:10.2106/JBJS.D.02536
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2671
COPYRIGHT © 2005
BY
THE JOURNAL
OF
BONE
AND JOINT
SURGERY, INCORPORATED
Chitosan-Glycerol
Phosphate/Blood Implants
Improve Hyaline Cartilage
Repair in Ovine
Microfracture Defects
BY CAROLINE D. HOEMANN, PHD, MARK HURTIG, DVM, EVGENY ROSSOMACHA, MD, PHD, JUN SUN, MD, MSC,
ANIK CHEVRIER, PHD, MATTHEW S. SHIVE, PHD, AND MICHAEL D. BUSCHMANN, PHD
Investigation performed at Ecole Polytechnique, Montréal, Quebec, Canada;
University of Guelph, Guelph, Ontario, Canada; and BioSyntech, Laval, Quebec, Canada
Background: Microfracture is a surgical procedure that is used to treat focal articular cartilage defects. Although joint
function improves following microfracture, the procedure elicits incomplete repair. As blood clot formation in the microfracture defect is an essential initiating event in microfracture therapy, we hypothesized that the repair would be improved if the microfracture defect were filled with a blood clot that was stabilized by the incorporation of a thrombogenic
and adhesive polymer, specifically, chitosan. The objectives of the present study were to evaluate (1) blood clot adhesion in fresh microfracture defects and (2) the quality of the repair, at six months postoperatively, of microfracture defects that had been treated with or without chitosan-glycerol phosphate/blood clot implants, using a sheep model.
Methods: In eighteen sheep, two 1-cm2 full-thickness chondral defects were created in the distal part of the femur
and treated with microfracture; one defect was made in the medial femoral condyle, and the other defect was made
in the trochlea. In four sheep, microfracture defects were created bilaterally; the microfracture defects in one knee received no further treatment, and the microfracture defects in the contralateral knee were filled with chitosan-glycerol
phosphate/autologous whole blood and the implants were allowed to solidify. Fresh defects in these four sheep were
collected at one hour postoperatively to compare the retention of the chitosan-glycerol phosphate/blood clot with that
of the normal clot and to define the histologic characteristics of these fresh defects. In the other fourteen sheep, microfracture defects were made in only one knee and either were left untreated (control group; six sheep) or were
treated with chitosan-glycerol phosphate/blood implant (treatment group; eight sheep), and the quality of repair was
assessed histologically, histomorphometrically, and biochemically at six months postoperatively.
Results: In the defects that were examined one hour postoperatively, chitosan-glycerol phosphate/blood clots showed
increased adhesion to the walls of the defects as compared with the blood clots in the untreated microfracture defects.
After histological processing, all blood clots in the control microfracture defects had been lost, whereas chitosanglycerol phosphate/blood clot adhered to and was partly retained on the surfaces of the defect. At six months, defects
that had been treated with chitosan-glycerol phosphate/blood were filled with significantly more hyaline repair tissue
(p < 0.05) compared with control defects. Repair tissue from medial femoral condyle defects that had been treated with
chitosan-glycerol phosphate/blood contained more cells and more collagen compared with control defects and showed
complete restoration of glycosaminoglycan levels.
Conclusions: Solidification of a chitosan-glycerol phosphate/blood implant in microfracture defects improved cartilage repair compared with microfracture alone by increasing the amount of tissue and improving its biochemical composition and cellular organization.
Clinical Relevance: The use of chitosan-glycerol phosphate/blood implants in conjunction with microfracture can improve the structural and compositional properties of repaired cartilage. These effects may result in better integration,
improved biomechanical properties, and longer durability of the repair tissue.
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S
urgical resurfacing techniques have been used for the treatment of focal articular lesions, osteochondritis dissecans,
and degenerative cartilage lesions1-7. For most patients,
pain and function scores improve for several years after microfracture or drilling3,5,7,8, but full recovery of joint function and
complete relief of pain are not always attained. Furthermore,
some patients simply fail to respond to this approach1,5,7-9. This
may be because these procedures create repair tissue that consists
of a mixture of fibrocartilage and hyaline cartilage3,5,6,9 that is not
attached to or integrated with the surrounding cartilage and thus
is subject to reinjury and rapid degeneration9. Despite promising
results in previous studies2,5, some patients are obliged to undergo additional surgical treatment, suggesting that improvements in standard surgical resurfacing therapies are needed.
During the microfracture procedure, a physical conduit is
created to connect vascularized subchondral bone marrow
with the débrided cartilage lesion. Animal models involving
rabbits10-12, dogs13,14, and horses15-21 have been developed to study
the repair tissue that is formed following resurfacing procedures. Taken together, these studies have indicated that, over the
course of three to twelve months, there is partial filling of these
defects with mainly fibrous repair tissue, with corresponding
control defects demonstrating considerably less filling. All of
these procedures require the formation of a blood clot and an
inflammatory response that stimulates bone marrow cells to
migrate, proliferate, and form a granulation tissue, which is
gradually replaced with fibrocartilage or fibrous repair tissue12,20.
The repair tissue should become integrated with the surrounding cartilage and should form a seamless junction with the articular surface in order to persist under load.
Since the first step in marrow stimulation is the formation of a blood clot, stabilization of the clot by incorporating a
thrombogenic polymer scaffold might improve the ensuing repair response. Chitosan is a particularly promising scaffolding
material because this polysaccharide, which is composed
predominantly of polyglucosamine, is positively charged and
thrombogenic22-24. Chitosan is prepared by deacetylation of
chitin, a natural polymer composed of acetylated glucosamine.
The polycationic nature of chitosan facilitates adhesion to negatively charged surfaces of tissues and cells25, including cartilage.
A wide range of medical uses for chitosan have been discovered
in the past fifteen years, including accelerated skin and fracture
repair26,27. Other studies have demonstrated biocompatibility,
low toxicity, and negligible immunogenicity23,28,29. A cytocompatible chitosan solution, chitosan-glycerol phosphate, also has
been generated by using glycerol phosphate as a buffer to titrate
acid-soluble chitosan to near-neutral pH and isotonic osmolarity30. For the current study, these chitosan-glycerol phosphate solutions were combined with sheep peripheral whole
blood to form a thrombogenic mixture that solidified and adhered to a full-thickness cartilage defect. We hypothesized that
this chitosan-glycerol phosphate/blood mixture could be applied to articular cartilage defects with microfracture holes to
stabilize the blood clot and to elicit an improved repair process.
We tested this hypothesis in sheep by creating defects in two locations at which focal defects frequently occur in patients.
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
Materials and Methods
Chitosan-Glycerol Phosphate/Blood Implants
edical-grade chitosan with a 79% degree of deacetylation31 was obtained from BioSyntech (Laval, Quebec,
Canada). Chitosan was dissolved at 1.89% weight per volume
in 70 mM HCl, autoclave-sterilized, and combined with filtersterile disodium beta-glycerol phosphate to yield a liquid chitosan-glycerol phosphate solution (also called BST-CarGel)
consisting of 1.7% weight-per-volume chitosan/135 mM glycerol phosphate (pH 6.8). Chitosan-glycerol phosphate/blood
mixtures were prepared in 4-mL glass screw-cap vials containing 1.1 mL of chitosan-GP (thawed from storage at 80°C), 3.3
mL of fresh sterile autologous whole blood drawn from the
jugular vein, and six 0.39-g surgical steel mixing beads (Salem
Specialty Ball, Canton, Connecticut). The contents of the vials
were mixed by means of ten seconds of vigorous shaking. The
surgeon then aseptically transferred the liquid mixture from
the vial to the surgical defect, within two minutes after mixing, with use of a 3-mL syringe and 20-gauge needle. Each implant was allowed to solidify in situ for seven to ten minutes.
Each condylar and trochlear defect required a separate autologous blood sample to be drawn for each chitosan-glycerol
phosphate/blood mixture.
M
Sheep Cartilage Repair Model
All procedures were approved by institutional animal care
committees according to Canadian Council on Animal Care
guidelines. Eighteen adult female sheep, all of which were retired Suffolk-Dorset breeders weighing 70 to 90 kg and ranging in age from three to six years, were selected from specific
pathogen-free herds that had been managed with a health
program that included vaccination against infectious diseases, antiparasiticides, and nutrition and infectious disease
surveillance. Sheep were allocated to groups by means of
blinded choice of eartag numbers. Sheep were anesthetized
with intravenous ketamine and diazepam and then were
maintained on halothane gas prior to lateral parapatellar arthrotomy. A surgical chisel and curet were used to remove
articular cartilage, with the maintenance of perpendicular
cartilage side-walls, to generate 1-cm2 full-thickness chondral defects. Previous studies have demonstrated that such
defects fail to repair spontaneously15,17.
In the present study, we aimed to remove as much calcified cartilage as possible without damaging the subchondral
bone. In all eighteen sheep, cartilage defects were created in either one knee or both knees (as described below). In all knees
with operatively created defects, one defect (9 × 10.5 mm) was
made in the central load-bearing region of the medial femoral
condyle and one defect (7.6 × 12 mm) was made in the lateral
distal facet of the trochlea (Table I).
In fourteen sheep, cartilage defects were created in
only one knee and the contralateral knee was left intact in
order to allow for the comparative measurement of thickness, safranin-O staining, and biochemical composition between the repaired cartilage and normal cartilage in the
regions of the defects (see schematic, Table IV). In each knee
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C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
TABLE I Animal Assignment and Treatment
Animal Number
Operated
Stifle
3-Week
Prior Impact
Chitosan-Glycerol
Phosphate/Blood
Clot Treatment
Repair
Period (wk)
Condylar Defect
Subchondral
Cyst Score*†
Fresh defects
23
L+R
—
Yes, L
0
NA
24
L+R
—
Yes, R
0
NA
25
26
L+R
L+R
—
—
Yes, L
Yes, R
0
0
NA
NA
12
13
L
R
—
—
—
—
29
29
2.5
0
21
22
R
L
—
—
—
—
27
27
3.5
1.5
15
L
Yes
—
28
1
16
L
Yes
—
28
1
8
9
R
L
—
—
Yes
Yes
30
30
0
0.5
17
18
R
L
—
—
Yes
Yes
27
27
2.5
1
19
20
R
L
—
—
Yes
Yes
27
27
2
0
6-month repair
3
R
Yes
Yes
30
0.5
5
R
Yes
Yes
30
0
*Scale = 0 (no cyst) to 4 (severe cyst). †NA = not applicable.
that had been operated on, both defects were treated either
with microfracture only (control group; six sheep) or with
microfracture and chitosan-glycerol phosphate/blood (treatment group; eight sheep), followed by a six-month repair period (Table I). Two animals from each six-month repair
group were administered a contusive impact injury (30 MPa;
impulse duration, 10 msec) in the medial femoral condyle
three weeks before creation of the defects. Such impact injuries induce immediate cartilage degeneration and eventually
progress to osteoarthritis over six months, as reported
previously32. With use of an arthroscopic microfracture awl
and mallet, fourteen to twenty evenly spaced microfracture
holes (measuring 1.5 mm in diameter and 3 mm deep) were
made in each defect, starting with a ring of holes spaced 2 to
3 mm apart at the defect rim and followed by five to eight
holes in the center. The open joint and soft-tissue surfaces
were continually rinsed with Ringer lactate solution. The
knee was closed in three separate layers with resorbable
suture material. Postoperatively, all animals received nonsteroidal antiinflammatory pain medication (75 mg of intravenous flunixin meglumine; Bimeda-MTC Animal Health,
Cambridge, Ontario, Canada) and antibiotics (20,000 IU/kg
of penicillin G; Novopharm, Toronto, Ontario, Canada) for
three days, followed by intermittent administration of antiinflammatory medication for as long as two weeks. All animals were walking within an hour after recovery from
anesthesia. Postoperative mobility was partly limited by re-
ducing pen size for one week. Temporary postoperative
lameness was observed in all sheep for one to three weeks after the arthrotomy.
In the cases of the remaining four sheep, bilateral cartilage defects were created, the joints were sutured, and the animals were killed immediately postoperatively in order to
characterize the initial cartilage defects and the retention of
implant in the defects. Defects in alternating left or right knees
were treated with chitosan-glycerol phosphate/blood implants (Table I). These four sheep were killed on the operating
table within one hour after the creation of the defects on the
side without implants and within 2.5 hours after the creation
of the defects on the side with implants.
At the time when the animals were euthanized, lameness, joint diameter (as an indication of effusion), hematological studies, and synovial fluid analysis were used to
evaluate safety and biocompatibility. During necropsy, all
joints were kept moist with Ringer lactate solution. Defect
sites were harvested with a band saw and were photographed
with a digital camera and dissection microscope. Full-thickness
biopsy specimens of repair tissue weighing 1 to 10 mg were
recovered from the distal medial corner of each trochlear
and condylar defect, from proximal sites outside of the defects, and from four matching sites from the intact, contralateral knee (see schematic, Table IV). Biopsy specimens
were weighed, frozen in liquid nitrogen, and stored at 80°C
for biochemical analysis.
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Sample Processing, Histomorphometric Analysis,
and Immunohistochemical Analysis
All bone-cartilage samples were fixed in formalin for five to
seven days and were decalcified in 0.5 N HCl with 0.1% glutaraldehyde for as long as two weeks. Repaired defects were trisected transversely into three blocks of equal size, postfixed, and
embedded in paraffin to obtain two distinct, 6-µm-thick osteochondral sections that included the entire width of the defect (7
to 10 mm) and 2 mm of flanking cartilage. In these two sections, the cross-sectional area of the repair tissue was defined as
all nonmineralized tissue above the subchondral bone plate, excluding occasional soft tissue in remnant microfracture holes.
One safranin-O-stained section was also prepared from a matching region in the intact, contralateral joint and was used to measure the cartilage thickness, calcified layer thickness, safranin-O
staining, and the cross-sectional area of the normal cartilage in
the regions of the defects. Histomorphometric analysis was performed on two safranin O/fast green-stained sections per defect by two independent, blinded observers (C.D.H. and A.C.)
with use of low-magnification digital images, generated with a
Zeiss dissection microscope and a digital camera and analyzed
with histomorphometry software (Northern Eclipse; Empix,
Mississauga, Ontario, Canada), to determine percent fill and
the percentage of hyaline cartilage in the repair tissue. Percent
fill was determined by measuring the cross-sectional area of the
repair tissue in a defect and then dividing this value by the
cross-sectional area of the normal cartilage in the same region
of the contralateral, intact joint. To estimate the amount of
hyaline cartilage in the repair tissue, it was assumed that tissue
that stained pink or red with safranin O was hyaline cartilage.
The percentage of the repair tissue that was hyaline cartilage was
then determined as the percentage of the total repair tissue area.
Calibrated line measurements were used to determine the thickness of the cartilage, calcified cartilage, and repair cartilage. For
fresh defects analyzed one to two hours after their creation, two
adjacent sections were generated from the middle of each defect
and were used to determine the percentage of the cross-section
of the defect base that was composed of noncalcified cartilage,
calcified cartilage, bone, or microfracture holes and to determine the thickness of noncalcified and calcified cartilage in the
flanking regions outside the defects.
For immunohistochemical analysis, sections were dewaxed and treated successively with trypsin for collagen type I
or pronase for collagen type II, followed by hyaluronidase33
and antigen retrieval34. Sections were then blocked with 20%
goat serum in PBS/0.1% Triton X-100, followed by successive
incubations with 1:50 dilution mouse monoclonal antibodies
to human collagen type II (II-4C11) or type I (I-8H5) (ICN
Biochemicals, Costa Mesa, California), biotin-conjugated goat
anti-mouse, and ABC-Alkaline Phosphatase Red Substrate
Detection kit (Vectastain; Vector Laboratories, Burlingame,
California). Controls, with secondary antibody only, showed
no background staining.
International Cartilage Repair Society (ICRS) histological profiles were generated with use of a modification of the system described by Mainil-Varlet et al.35. The repair tissues
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
contained within the defects were scored as four contiguous,
approximately 2-mm segments. Each 2-mm segment was individually scored for six different parameters, including (I) surface smoothness (with 3 indicating smooth and 0 indicating
irregular), (II) the type of matrix (with 3 indicating hyaline and
0 indicating fibrous), (III) cell organization (with 3 indicating
columnar and 0 indicating random), (IV) cell viability (with 3
indicating predominantly viable cells and 0 indicating few viable cells), (V) the absence of subchondral bone pathology (with
3 indicating normal, 2 indicating remodeled, and 1 or 0 indicating detached and/or necrotic), and (VI) calcified cartilage (with
3 indicating normal and 0 indicating abnormal [formed >100
µm higher or lower than the adjacent tidemark] ). The final
scores for each parameter were the collective average from two
sections per defect and four segments per section as evaluated
by two independent blinded observers (A.C. and E.R.).
Determination of Collagen Content,
Glycosaminoglycan Content, and Cell Density
Thawed cartilage samples were weighed and subjected to papain digestion at pH 6.5 to determine the DNA content (by
means of Hoechst fluorometric assay), glycosaminoglycan content (by means of DMB assay), and collagen content (by means
of hydroxyproline assay), normalized to wet weight36. Samples
for biochemical analysis were not retrieved from the defect
when repair tissue was absent from the distal medial corner.
Cell density was determined with use of 7.7 pg DNA/cell37, and
collagen content was determined with use of 7.6 mg collagen/
mg hydroxyproline38,39. Standards included sheared calf thymus
DNA, 4-trans-hydroxyproline, and shark cartilage chondroitin
sulfate B (Sigma-Aldrich, Oakville, Ontario, Canada).
Safety Outcome Measures
Peripheral EDTA-anticoagulated blood and synovial fluid were
collected at the time of surgery and at the time of necropsy for
hematological analysis and for synovial fluid analysis of total serum protein, mucin, and leukocyte counts (University of
Guelph Animal Health Laboratory, Guelph, Ontario, Canada).
Lameness was evaluated on a scale of 0 (no lameness) to 5 (severe lameness). Joint effusion was evaluated by comparing the
diameters of stifles with and without operatively created defects
and by measuring the total synovial fluid aspirated at the time
of necropsy. Subchondral cysts were scored histomorphometrically for both sections taken from within each defect, and the
scores were averaged. Cyst severity, as indicated by the diameter
of fibrous white tissue or tissue voids, was evaluated on a scale
of 0 (<1 mm) to 4 (>6 mm).
Statistical Analysis
The effects of treatment (microfracture alone or microfracture
combined with chitosan-glycerol phosphate/blood) and defect
location on percent fill in repair cartilage were analyzed with
use of analysis of variance with repeated measures, with each
of the two sections analyzed by each of the two blinded observers constituting the four repeated measures (STATISTICA,
version 6.1; StatSoft, Tulsa, Oklahoma). Percent hyaline con-
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H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
TABLE II Histomorphometric Analysis of Defects and Contralateral Intact Cartilage
Medial Femoral Condyle*†
Control
(N = 4)
Treated
(N = 4)
Lateral Trochlea*†
Control
(N = 4)
Treated
(N = 4)
Fresh defects
Outside the defects
Calcified cartilage thickness (µm)
Noncalcified cartilage minimum thickness (µm)
181 (116)
328 (165)
177 (112)
346 (98)
127 (58)
499 (209)
141 (70)
471 (198)
Noncalcified cartilage maximum thickness (µm)
1074 (224)
957 (369)
638 (124)
763 (249)
Débrided calcified cartilage thickness (µm)
Percent calcified cartilage‡
153 (119)
60% (28%)
156 (113)
48% (14%)
104 (82)
55% (15%)
106 (89)
53% (17%)
Percent microfracture hole‡
Percent bone exposed‡
21% (10%)
14% (19%)
22% (10%)
13% (13%)
20% (4%)
16% (7%)
20% (9%)
21% (15%)
Percent noncalcified cartilage‡
Percent macroscopic clot covering defect
5% (6%)
27% (25%)
15% (11%)
41% (27%)
8% (15%)
69% (23%)
5% (7%)
80% (32%)
Inside the defects
Control
(N = 6)
Treated
(N = 8)
Control
(N = 6)
Treated
(N = 8)
Six-month postoperative repair
Unoperated joints
Calcified cartilage thickness (µm)
171 (67)
184 (118)
223 (128)
221 (109)
Noncalcified cartilage minimum thickness (µm)
Noncalcified cartilage maximum thickness (µm)
390 (36)
1520 (260)
424 (108)
1316 (268)
643 (154)
575 (65)
564 (113)
580 (76)
Percent hyaline cartilage
100%
100%
94% (9%)
85% (18%)
Repair tissue
Percent hyaline cartilage in repair tissue
71% (11%)#
86% (7%)#**
43% (20%)
59% (21%)††
Percent fill in defect§
Minimum thickness of repair tissue (µm)
31% (7%)
55 (130)
52% (21%)‡‡
176 (180)
69% (23%)
321 (282)
94% (40%)§§
311 (290)
Maximum thickness of repair tissue (µm)
Percent residual calcified cartilage‡
538 (173)
24% (26%)
645 (172)
9% (14%)
706 (301)
ND##
918 (327)
ND##
Percent residual noncalcified cartilage‡
ND##
ND##
Percent collagen type II repair
99% (2%)
9% (15%)
96% (8%)
6% (12%)
71% (15%)
87% (13%)
Percent collagen type I repair
2% (4%)
3% (3%)
19% (12%)
16% (15%)
*The data are given as the mean, with the standard deviation in parentheses. †Control = microfracture only; treated = microfracture and chitosan-glycerol phosphate/blood implant. ‡Percentage of the cross-section of the defect. §Percentage of the repair tissue cross-sectional
area divided by the area of the cartilage in the matching location in the intact joint. #p < 0.05 compared with trochlear defects. **p < 0.05
compared with untreated defects. ††p = 0.066 compared with untreated defects. ‡‡p = 0.062 compared with untreated defects. §§p =
0.093 compared with untreated defects. ##ND = not done.
tent in repair tissue was statistically analyzed in an identical
fashion to that used for percent fill. Averaged data from one
observer differed from that of the other observer by <3% for
both percent fill and percent hyaline repair for all sections, indicating the reproducibility of this histomorphometric procedure. Percent fill and percent hyaline were also analyzed
together as an aggregate indicator of the overall quantity of
hyaline repair cartilage by specifying two repeated-measure
variables, one being either percent fill or percent hyaline and
the other being the section number (1 to 4), for multivariate
analysis of variance (STATISTICA GLM; StatSoft). International Cartilage Repair Society histological scores assigned by
two independent observers to two sections per defect were an-
alyzed with use of analysis of variance with four repeated measures to examine the effect of location (trochlea or condyle) as
well as that of chitosan-glycerol phosphate/blood treatment
on International Cartilage Repair Society repair parameters.
Cyst scores and biochemical data (glycosaminoglycan content,
collagen content, and cell number) were analyzed with use of
the Student t test. The level of significance was set at p < 0.05.
Results
Characterization of the Fresh Defects and
Chitosan-Glycerol Phosphate/Blood Implant Retention
uring the clinical microfracture surgical procedure, it is
normally recommended that the zone of calcified carti-
D
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lage be removed without damaging the subchondral bone1.
Therefore, in the present study, we aimed to remove the calcified cartilage without damaging the subchondral bone. Histological analysis of the fresh defects collected from four sheep
one hour postoperatively revealed that it is difficult to consis-
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
tently remove the entire zone of calcified cartilage, even when
the microfracture procedure is carefully carried out. In the
fresh treated or control defects, a similar average of 48% to
60% of the cross-sectional area of the calcified cartilage still
remained, and the thickness of the remaining calcified carti-
Fig. 1
Photographs of initial defects (A through F) and defects collected as long as one hour (H, K, L) or as long as 2.5 hours (G, I, J) after microfracture
and in situ solidification of a chitosan-glycerol phosphate/blood implant. A and D: Lateral trochlear defect (A) and medial condylar defect (D) immediately after microfracture and rinsing with Ringer lactate solution. B and E: Punctuate bleeding was observed five minutes later in trochlear (B) and
condylar (E) defects. C and F: Chitosan-glycerol phosphate/blood implant solidified in situ in trochlear (C) and condylar (F) defects. G and H: Fresh
defects from an animal killed 1.5 hours after implantation of chitosan-glycerol phosphate/blood (G) and one hour after generation of the control defect (H). I through L: Decalcified and trimmed specimens showing chitosan-glycerol phosphate/blood clot solidified in defects from the trochlea (I)
and condyle (J) and spontaneous bleeding in control defects from the trochlea (K) and condyle (L). The small black arrows in I through L indicate microfracture holes filled with blood.
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lage varied considerably (Table II). Some residual noncalcified
articular cartilage also remained (Table II), mainly at the edges
of the fresh defects. Microfracture holes penetrated 2.5 to 3
mm into the subchondral bone plate. Except for the micro-
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
fracture holes, the subchondral bone was intact.
All microfracture holes filled slowly with blood over a
period of five minutes (Fig. 1, A and D compared with B and
E). Approximately fifteen minutes after the generation of mi-
Fig. 2
Histological appearance of paraffin-embedded sections obtained from freshly prepared defects collected one to 2.5 hours after defect generation.
A and B: Low-magnification images of a section from the intact contralateral trochlear (A) or condylar (B) defect region. C and D: The middle of an initially created defect in the trochlea (C) or condyle (D) bisecting a microfracture hole (black arrow). Note that all normal blood clot was lost during paraffin histoprocessing, as previously observed12,41,42. E through H: Control defects failed to retain any clot material in paraffin-embedded sections
from the trochlea (E) or condyle (G), whereas defects with chitosan-glycerol phosphate/blood implant formed an adhesive bond between calcified
cartilage and the implant in the trochlea (F) and condyle (H) that remained intact even after histoprocessing. Open arrowheads indicate chitosan
stained with fast-green56, and black arrowheads indicate clotted erythrocytes.
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C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
Fig. 3
Condylar defects at six months postoperatively. A1 through B4: Control (microfracture-only) defects from animals 16 and 22. C1 through D4: Defects treated with microfracture and chitosan-glycerol phosphate/blood from animals 3 and 9. Defects are shown for condyles with a previous threeweek antecedent impact injury (A1 through A4 and C1 through C4) or from healthy stifles (B1 through B4 and D1 through D4). A1, B1, C1, and D1:
Macroscopic appearance immediately after microfracture and after delivery of chitosan-glycerol phosphate/blood (C1 and D1). A2, B2, C2, and D2:
Macroscopic appearance after six months of repair, with two distinct sections generated from each defect (arrow and double arrow). A3, B3, C3, D3,
A4, B4, C4, and D4: Safranin-O/fast-green-stained paraffin sections. Arrowheads show defect boundaries, and dotted circles show the perimeter of
subchondral cysts or subchondral fibrous tissue. A4: Open arrowhead shows a region of calcified cartilage not covered by repair tissue. B2 and B4:
Open arrow shows the cleft communicating with a subchondral cyst. C2: Dotted circle encompasses the biochemistry sample site.
crofracture holes, a dark bruise could be seen forming underneath the defects, indicating that subchondral bleeding was
occurring between the microfracture holes. This bruise also
was apparent in trimmed decalcified defects as a general discoloration of subchondral tissues (Fig. 1, I through L).
In all treated defects, a fresh mixture of chitosan-glycerol phosphate solution and peripheral whole blood was applied as a viscous liquid, which solidified in situ within seven
to ten minutes (Fig. 1, C and F; Fig. 3, C1 and D1; and Fig. 4,
C1 and D1). Fat droplets could be seen forming on the surface
of chitosan-glycerol phosphate/blood before solidification.
When two joints were cycled through a complete range of motion eight times, an adhesive layer of the implant remained in
the defects. At the time of necropsy, a variable but higher average percentage of the defect surface was covered with chitosanglycerol phosphate/blood implant as compared with normal
clot in both condylar and trochlear defects (Table II and Fig. 1,
G and H). More clot was present in fresh trochlear defects,
which faced a smooth patellar surface, as compared with
condylar defects facing the meniscus, tibial plateau, and fat
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pad, which is quite prominent in the sheep knee40. In one
control condylar defect that had been treated with microfracture only, the clot was limited to the microfracture holes
(Fig. 1, H).
Evaluation of paraffin-embedded sections of these fresh
defects revealed that all normal blood clot material had been
lost during histoprocessing (Fig. 2, C, D, E, and G), which is
consistent with the findings reported by previous investigators12,41,42. However, chitosan-glycerol phosphate/blood adhered tightly to the calcified cartilage and bone and was partly
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
retained on the surface of treated defects after histoprocessing
(Fig. 2, F and H, open arrowhead).
Histological and Biochemical Evaluation
of Six-Month Repair Tissues
After six months of repair, all condylar repair tissue was glossy,
white or grey, and firmly attached (Fig. 3). Some condylar repair tissues contained a cleft communicating with a subchondral cyst (Fig. 3, B2, open arrow). Treated condylar defects
were macroscopically resurfaced with more repair tissue. New
Fig. 4
Representative trochlear defects and corresponding six-month repair tissues. A1 through B4: Control (microfracture-only) defects from animals 16
and 22. C1 through D4: Defects treated with microfracture and chitosan-glycerol phosphate/blood from animals 3 and 20. Trochlear defects were
created in animals with an impact injury to the condyle made three weeks before this surgery (A1 through A4 and C1 through C4) or in healthy stifles
(B1 through B4 and D1 through D4). A1, B1, C1, and D1: Macroscopic appearance immediately after microfracture and after delivery of chitosanglycerol phosphate/blood (C1 and D1). A2, B2, C2, and D2: Macroscopic appearance after six months of repair showing the location of sections (arrow and double arrow). A3, B3, C3, D3, A4, B4, C4, and D4: Safranin-O/fast-green-stained sections of each trimmed plane in the defect. Arrowheads
show original defect boundaries. White arrowheads indicate less safranin O-stained repair tissue in distal as compared with proximal regions in trochlear defects.
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C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
Fig. 5
Histologic appearance of safranin O/fast-green-stained repair cartilage after six months of repair. Example repair tissues from treated (A, C, and E)
and control (B, D, and F) defects. More hyaline repair was seen in the condyle (A and B) than in the trochlea (C through F). Distal trochlear repair tissues closest to the intercondylar notch were consistently glycosaminoglycan-depleted (E and F). The arrow in B indicates tidemark formed below columnar repair cartilage. (Scale bar = 100 µm.)
repair tissue had failed to cover residual cartilage, either calcified or noncalcified. The percentage of the defects covered
with residual calcified cartilage was 9% in treated condylar defects compared with 24% in control defects (Table II). The
amount of residual noncalcified cartilage in treated condylar
defects was similar to that of controls (percentage covered, 6%
in treated and 9% in controls) (Table II). Compared with microfracture-only defects, treated condylar defects were filled
with a greater cross-sectional area of repair tissue (percent fill,
52% in treated and 31% in controls) (Table II) and demonstrated a significantly higher percentage of hyaline repair
(86% in treated and 71% in controls) (Table II), which, when
taken together, resulted in a significant increase in combined
percent fill and percent hyaline repair over microfracture-only
controls (p < 0.05). The average thickness of the repair tissue
was greater in treated than in untreated defects but only attained half the thickness of the articular cartilage in the con-
tralateral, intact joints (Table II). According to a modification
of the International Cartilage Repair Society histological scoring system35 (as described in the Methods section), repair tissue from treated condylar defects had a smoother surface,
with more columnar organization and significantly more hyaline character (p < 0.05) (Table III, Fig. 5). When columnar
repair cartilage was present, a new tidemark was often found
at the base (Fig. 5, B, arrow). Interestingly, all condylar repair
tissue, whether from treated or untreated defects, stained positively for collagen type II, encompassing approximately 97%
of the total cross-sectional area from the cartilage-bone interface to the most superficial region of the repair tissue (Table II;
Fig. 6, B, G, and H). Only a negligible amount (approximately
3%) of condylar repair tissue stained with anti-collagen type I,
which uniformly stained the subchondral bone (Fig. 6, C and
I). A more porous subchondral bone was found below repair
tissue in treated defects (Fig. 6, G and I). Treated defects gen-
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erally showed improved repair tissue integration, which also
depended on percent fill (Fig. 3), although many repair tissues
were still not fully integrated with the adjacent articular cartilage after six months of repair.
Repair tissue biopsy specimens from treated condylar
defects contained nearly twofold more glycosaminoglycan,
collagen, and cells compared with biopsy specimens from
control defects treated with microfracture alone (Table IV, site
4). These treatment-dependent higher glycosaminoglycan and
collagen levels were nearly significant (p ≅ 0.06), despite the
low sample number from the control group (three of six) due
to the fact that half of the control defects were devoid of repair
tissue at biopsy site 4. Both treated and control repair tissues
had an identical average glycosaminoglycan/cell ratio of 0.9
ng/cell (Table IV, site 4).
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
In intact, contralateral joints, significant site-specific
biochemical differences were found. The central part of the
medial femoral condyle had the highest glycosaminoglycan/
cell ratio (3.0 ng/cell), and the distal part of the trochlea had
the lowest glycosaminoglycan/cell ratio (0.5 ng/cell) (Table IV,
sites 4 and 2, respectively). Repair tissue from treated condylar
defects contained significantly higher cell densities (57 compared with 16 million cells/g wet weight), increased collagen
per wet mass (140 compared with 111 mg/g wet weight), and
similar glycosaminoglycan content (49 compared with 48 mg/
g wet weight) compared with articular cartilage from matching sites in intact joints (Table IV, site 4).
Trochlear repair tissue was macroscopically heterogeneous compared with condylar repair tissue, with either cobblestone tufts over the microfracture holes, a dense white or
Fig. 6
Immunohistochemistry analysis of collagen type II and collagen type I in six-month repair tissue in chitosan-glycerol phosphate/blood-treated defects
from animal 20 (A through F) and animal 9 (G, H, and I). A, B, C, G, and H: Condylar repair tissue. D, E, F, and I: Trochlear repair tissue. A and D: Safranin
O-stained sections adjacent to those sections stained with monoclonal antibodies specific for collagen type II (B and E) and collagen type I (C and F).
Positive immunostaining was revealed by red substrate, with hematoxylin counterstain, resulting in a purple hue for collagen type-I staining in bone. The
arrowheads in A, B, C, D, E, F, and I delineate the original defect borders. The open arrow in F indicates red collagen type-I positive staining in trochlear
repair cartilage. G: In collagen-type-II-stained condylar repair tissue, columnar repair tissue originated from porous bone. H: The collagen-type-II-stained
fibers were often present as looped structures that originated from the bone and formed an acute intensely stained arc at the surface of the columnar
repair tissue. I: Collagen type I immunostain showing a seamless integration of repair tissue with trochlear articular cartilage and a seamless integration of bone below the treated defect with the subchondral bone plate. The black arrowhead indicates the junction of repair cartilage (RC) and adjacent
cartilage at the original tidemark, and the double-headed arrow indicates calcified cartilage thickness in adjacent cartilage.
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C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
TABLE III International Cartilage Repair Society Histological Scoring of Six-Month Postoperative Repair
Medial Femoral Condyle
Control*
(N = 6)
Treated†
(N = 8)
Lateral Trochlea
Control*
(N = 6)
Treated†
(N = 8)
2.3
2.7
Criterion
I Surface (3 = smooth, 0 = irregular)
1.6
2.0
II Matrix (3 = hyaline, 0 = fibrous)
1.7‡
2.1§#
1.1
1.5§
III Cell organization (3 = columnar, 0 = random)
1.2**
1.7**
0.5
0.6
IV Viability (3 = viable, 1 = partially viable, 0 = few viable)
2.3
2.7
2.5
2.7
V Bone (3 = normal, 2 = remodeled, 1 and 0 = detached and/or necrotic)
1.6
1.8
2.0
2.0
VI Calcified cartilage (3 = normal, 0 = abnormally high/low)
2.2
2.0
1.0
1.0
*Control = microfracture only. †Treated = microfracture and chitosan-glycerol phosphate/blood implant. ‡p < 0.05 compared with trochlear defects. §p < 0.05 compared with untreated defects. #p < 0.01 compared with trochlear defects. **p < 0.0001 compared with trochlear defects.
whitish-grey covering, or a spongy white uniform covering
(Fig. 4, A2, B2, C2, and D2). Histomorphometrically, treated
defects had higher percent fill (94% compared with 69%),
mainly because some treated defects showed modest bone resorption of the defect base, which led to some defects having
>100% fill (Table II; Fig. 4, D4). In many trochlear defects, the
defect base was irregular with respect to the adjacent tidemark
(Table III, parameter VI). Compared with condylar repair tissue, trochlear repair tissue was significantly less hyaline on
histomorphometric analysis (p < 0.05) and was significantly
less hyaline (p < 0.05) and columnar (p < 0.0001) on histological analysis (Table III, parameters II and III). Consistent with
histological scoring for fibrocartilage, an appreciable amount
of collagen type I was detected in trochlear repair sections on
immunohistochemical analysis (16% to 19% of repair tissue
area) (Table II; Fig. 6, F). Fibrous trochlear repair was most
frequently observed close to the intercondylar notch (Fig. 4,
B4 and D4, white arrowhead). Biochemical analysis of trochlear repair tissue taken from near the notch revealed that
this tissue was consistent with fibrous tissue, with very high
cellularity, high levels of collagen, and significantly less glycosaminoglycan compared with condylar repair tissue (p <
0.01) (Table IV, site 2). A beneficial effect of treatment was
noted for the trochlear defects; specifically, trochlear repair
tissue in treated defects had a higher average percentage of
type-II collagen staining (87% as compared with 71% of the
repair tissue area) (Table II) and demonstrated a significant
increase in combined percent fill and percent hyaline repair as
compared with that in control defects that had been treated
with microfracture only (p < 0.05). Trochlear repair tissue integration was generally better than condylar repair tissue integration (Fig. 6, I).
Safety Outcome Measures
At the time of necropsy at six months, safety tests indicated
that treatment of defects with chitosan-glycerol phosphate/
blood did not give rise to any lasting lameness, effusion, or
systemic inflammation. A similar average amount of synovial
fluid (approximately 0.25 mL) was found in the joints with
treated or untreated defects. None of the sheep were lame at
six months, with the exception of one sheep in the treatment
group that had mild lameness. Subchondral cysts with varying
grades of severity were observed beneath all but one control
condylar defect (Fig. 3, A and B). In defects that had been
treated with chitosan-glycerol phosphate/blood, cysts were absent or much smaller (Table I; Fig 3, C and D). When evaluated on a scale of 0 to 4 (with 0 indicating no cyst and 4
indicating a large cyst), the severity of cysts was attenuated
50% by treatment (median, 0.5 compared with 1.25), but,
with the sample size used in the present study, this effect was
not significant. In operated joints, trochlear samples proximal
to the defects showed no biochemical differences compared
with matching unoperated sites (Table IV, site 1), ruling out
generalized joint degeneration. In contrast, condylar samples
proximal to the defects showed a selective and significant
(~20%) decrease in collagen content per wet weight in both
treated and control joints compared with matching unoperated sites (Table IV, site 3).
Discussion
revious cartilage repair studies in horses demonstrated
that carpal, trochlear, or condylar defects were partly
filled16,19,20 with repair tissues that were partly hyaline, with partial collagen type-II content and relatively low glycosaminoglycan levels20,43-46, after four to twelve months of repair.
Suboptimal repair in some of those studies potentially could
be attributed to the use of trochlear defects; our data indicated
that trochlear defects had significantly less hyaline repair and
glycosaminoglycan content compared with condylar defects,
results that are consistent with the findings reported by
others17,42. In the present study, condylar defects that had been
treated with microfracture alone were partly filled with repair
tissue (percent fill, 31%) that was partly hyaline (71%) and
had low glycosaminoglycan levels (27 mg/g wet weight). Application of a chitosan-glycerol phosphate/blood clot to condylar defects resulted in a net increase in fill (52%), a modest
P
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TABLE IV Biochemical Composition of Joint Tissue in Unoperated, Operated, and Repaired Sites
Site 1
Site 3
Proximal Trochlea
Proximal Condyle
Para-Defect
GAG‡§ (mg/g ww‡)
Collagen§ (mg/g ww#)
Cell density§ (million/g ww#)
ng GAG/cell‡
Para-Defect
Unoperated
(N = 12)
Control*
(N = 6)
Treated†
(N = 8)
Unoperated
(N = 12)
Control*
(N = 6)
Treated†
(N = 8)
45 (12)
40 (14)
39 (6)
34 (13)
30 (14)
35 (8)
122 (30)
124 (33)
131 (21)
151 (33)
115 (12)**
39 (18)
30 (4)
28 (3)
38 (11)
41 (22)
38 (8)
1.2
1.3
1.4
1.0
0.7
0.9
Site 2
Site 4
Distal Trochlea
Distal Condyle
Defect
GAGठ(mg/g ww#)
Collagen§ (mg/g ww#)
Cell density§ (million/g ww#)
ng GAG/cell‡
Unoperated
(N = 12)
Control*
(N = 5 or 6)
21 (12)††
18 (10)‡‡
145 (43)
53 (20)
0.5††
124 (8)**
Defect
Treated†
(N = 7)
11 (8)‡‡
Unoperated
(N = 12)
Control*
(N = 3)
Treated†
(N = 6)
48 (17)§§
27 (16)
49 (14)##
150 (22)
141 (45)
111 (36)§§
85 (33)
140 (34)***
123 (69)†††
160 (94)†††
16 (4)††
30 (13)
57 (27)†††
0.1**
0.1§§§
3.0††
0.9§§§
0.9**
*Control = microfracture only. †Treated = microfracture and chitosan-glycerol phosphate/blood implant. ‡GAG = glycosaminoglycan. §The
data are given as the mean, with the standard deviation in parentheses. #ww = wet weight. **p < 0.05 compared with the same site in the
unoperated joint. ††p < 0.005 compared with all other sites in the unoperated joint. ‡‡p < 0.01 compared with condylar repair tissue in the
same joint. §§p < 0.05 compared with the proximal site in the unoperated condyle. ##p = 0.063 compared with repair tissue from control
defects. ***p = 0.054 compared with repair tissue from control defects. †††p < 0.0001 compared with the same site in unoperated joints.
§§§p < 0.01 compared with the same site in unoperated joints.
but significant increase in hyaline character (86%), and a complete restoration of normal glycosaminoglycan levels (49 mg/g
wet weight) (Tables II, III, and IV). To our knowledge, this
high level of glycosaminoglycan content has never been reported in cartilage repair tissues from skeletally mature adult
large-animal models. Collagen type II was uniformly detected
throughout both treated and control condylar repair tissues,
and yet much less glycosaminoglycan was detected in repair
tissue in defects that had been treated with microfracture only.
This suggests that collagen type II expression is necessary, but
not sufficient to retain high glycosaminoglycan levels in repair
tissues. Our collective data suggest that the more cellular repair tissue (Table IV) with columnar organization (Table III)
elicited by chitosan-glycerol phosphate/blood clots in the condyle led to the deposition of more cartilage repair tissue (Table
IV) with greater hyaline character (Tables II, III, and IV), compared with microfracture alone.
Our biochemical analyses showed that collagen was selectively depleted in proximal condylar sites of joints with operatively created defects. High load-bearing on the defect edge
in the condyle may have led to damage and swelling of the collagen network, resulting in higher water content. These same
effects were not seen in the less load-bearing proximal trochlear sites. Trochlear defects were filled with more repair tissue than condylar defects were, but the repair tissue was much
more fibrous (Table III).
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Subchondral Cysts and Retention and
Stability of the Calcified Cartilage Layer
Subchondral cyst formation has been previously observed in
large-animal cartilage-repair models when the subchondral
plate was violated by débridement or drilling21,46, followed by
immediate postoperative weight-bearing47. A lack of bone replacement of the damaged subchondral plate also has been
seen as long as five to twelve months after surgery7,16,18,21,48.
Thus, although much available data suggest that percent fill
may be improved by a more exuberant débridement step18,21 in
these surgical procedures, one potential downside could be
that extensive damage to the subchondral plate, via excessive
débridement3 or communicating microfracture holes19, in addition to rapid postoperative weight-bearing47, could cause
pathological bone resorption and subchondral cyst formation.
Although not previously reported in animal or clinical
studies1,11,19,20, the subchondral bruising that we observed under
the microfractured defects also may be related to subchondral
cyst formation. Given that subchondral cysts have only rarely
been mentioned in the clinical literature as a complication of
drilling or microfracture49, we may assume that such cysts are
asymptomatic, undetected, resolve spontaneously, or simply
are not produced when controlled rehabilitation during the
first eight weeks postoperatively prevents full load-bearing. In
situ solidification of the chitosan-glycerol phosphate/blood
implant over the microfractured defects decreased the frequency and severity of these cysts (Table I). One possible
mechanism behind this treatment-induced suppression of cyst
formation may involve angiogenic22 or osteoconductive properties of chitosan50, which could facilitate a more effective repair of the subchondral bone damaged by microfracture.
Retention of the calcified layer and its persistence during cartilage repair has often proved to be mutually exclusive
with resurfacing and integration of repair tissue in cartilage
defects in dogs14,51 and horses16-19,21. The present study in sheep
demonstrated similar findings (for example, Fig. 3, A4, empty
arrowhead), suggesting that residual cartilage can be a barrier
to marrow-derived repair. In the present study, after six
months of repair, a comparable percentage of the defects was
covered with residual noncalcified cartilage in treated and
control defects (6% and 9%, respectively) compared with
fresh defects (10%) (Table II). This is in contrast with the residual calcified cartilage layer in the medial femoral condyle,
which was detected in fresh defects (percentage covered, 54%)
and to a lesser extent in control defects (24%) and treated defects (9%) (Table II). This observation, combined with the repair tissue that was seen to be uniformly adhering to bone at
six months (Fig. 3, C3 and D3, and Fig. 6, G and I), suggests
that the calcified cartilage can be resorbed during repair and
that chitosan-glycerol phosphate/blood implants increased resorption of residual calcified cartilage. Similar observations
have been previously made where implantation of slowly degrading biomaterials (such as collagen or polylactic acid fibers) into chondral defects can result in resorption of the
calcified layer and subchondral bone in dog and goat14,51,52.
Furthermore, recently published data suggest that implanta-
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
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tion of autologous cells also can result in a local resorption of
the calcified layer and subchondral bone in dogs42, goats53, and
human patients54, thereby solicting a marrow response.
Clinical Relevance and Limitations
of the Animal Model
Our model was designed to examine clinically relevant aspects
of cartilage repair, including different sites (i.e., the condyle and
trochlea) and the repair of posttraumatic lesions1,4,5. The entire
procedure of arthrotomy, defect creation, and application of the
implants took place within ninety minutes, a clinically acceptable time-frame. Terminal assessments were performed at six
months after microfracture, a time at which patients typically
are permitted full running and jumping activities, although
many patients only experience optimal clinical improvement
two years after microfracture2,8. Large-animal chondral defects
(area, 1 cm2; depth, 1.5 mm; volume, 150 µL) might be anticipated to heal in a manner closer to human defects as compared
with defects in small, inbred laboratory animals such as rabbits
(area, 0.02 cm2; depth, 100 µm; volume 2 µL). Also, we opted to
use skeletally mature sheep because we believed that they would
be more comparable to the patient population in need of treatment and because adolescent animals have a much greater potential for spontaneous repair55.
All animals were allowed immediate postoperative weightbearing, which had an unknown effect on implant retention.
No chitosan was detected in any of the sixteen treated defects
that were examined at six months, which is reasonable in light
of previous reports indicating that chitosan is readily degraded23 and cleared28. In blood and synovial fluid analyses
carried out at six months postoperatively, there was no evidence that the chitosan-glycerol phosphate/blood implant
caused any chronic inflammation. Collectively, these data suggest that chitosan-glycerol phosphate/blood implants exert an
efficacious effect on cartilage repair without any toxicity at
these dose levels. NOTE: The authors thank Deb McWade for animal husbandry, Julie Tremblay for quality assurance, and Drs. Pierre Ranger and Georges-Etienne Rivard for valuable discussions.
Caroline D. Hoemann, PhD
Anik Chevrier, PhD
Michael D. Buschmann, PhD
Department of Chemical Engineering and Institute of Biomedical Engineering, Ecole Polytechnique, P.O. Box 6079, Station Centre-Ville, Montréal, Quebec H3C 3A7, Canada. E-mail address for C.D. Hoemann:
[email protected]
Mark Hurtig, DVM
Comparative Orthopaedic Research Laboratory, Department of Clinical
Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario N1G 2W1, Canada
Evgeny Rossomacha, MD, PhD
Jun Sun, MD, MSc
Matthew S. Shive, PhD
BioSyntech Inc., 475 Armand-Frappier Boulevard, Laval, Quebec H7V
4B3, Canada
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In support of their research or preparation of this manuscript, one or
more of the authors received grants or outside funding from the Canadian Institutes of Health Research (CIHR) and the Canadian Arthritis
Network, of the Centres for Excellence. In addition, one or more of the
authors received payments or other benefits or a commitment or agreement to provide such benefits from a commercial entity (BioSyntech,
Inc.). No commercial entity paid or directed, or agreed to pay or direct,
C H I T O S A N -G L YC E RO L P H O S P H A T E /B L O O D I M P L A N T S I M P ROVE
H Y A L I N E C A R T I L A G E R E P A I R I N O V I N E M I C RO F R A C T U R E D E F E C T S
any benefits to any research fund, foundation, educational institution, or
other charitable or nonprofit organization with which the authors are affiliated or associated.
doi:10.2106/JBJS.D.02536
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