Keywords
microfracture - nanofracture - articular cartilage repair - mesenchymal stem cells
- sheep
Introduction
Although almost 60 years have passed since the description of the first bone marrow
stimulation technique by Smillie,[1] the treatment of chondral lesions so far represents a relevant and challenging issue.
Despite the fact that along the years and especially in the recent past several treatments
and novel biomaterials have been proposed, none of the different methods available
has been able to renovate the joint surface from both the biological and biomechanical
standpoint.[2] The microfracture technique developed by Steadman still represents the most frequently
used procedure.[3] Approximately 80,000 microfracture procedures are performed every year in the United
States.[4]
[5] The biological rationale of bone marrow stimulation is attributed to the role of
mesenchymal stem cells (MSCs) recruited from the bone marrow.[6]
[7]
[8]
[9]
[10]
[11] The advantages of this technique include its minimal invasiveness, favorable cost-effectiveness
ratio, and low technical demands. Satisfactory results are being achieved especially
in young patients with defects of limited size.[8]
[12]
[13] However, the quality and characteristics of the newly formed fibrocartilaginous
tissue have been widely discussed in the literature[14]
[15] due to the limited biomechanical properties of the tissue rich in type I collagen
and due to its tendency to degenerate.
In recent years, improved knowledge and understanding of the structural and functional
architecture of subchondral bone has led to an increased focus on the need to preserve
its integrity during chondral defect treatment.
The limited penetration and the broad diameter of the awl[16] are currently thought to play a key role in repairing fibrocartilaginous tissue,
as well as in subchondral bone compaction around the perforation[9]
[16]
[17] and in intralesional osteophytes formation.[9]
[18] To overcome the mechanical limitations of microfracture, the nanofracture technique
was recently introduced using smaller diameter and deeper subchondral bone needle
perforations (Arthrosurface Inc., Franklin, Massachusetts, United States).[9]
The aim of this study was to assess macroscopic, histological, and immunohistochemical
characteristics of repair tissue of chondral defects treated with microfractures or
nanofractures in an ovine model. The hypothesis of the study was that nanofracture
provides better repair tissue than microfracture.
Methods
The local ethic committee approved this study, and all procedures were conducted according
to the Institutional Animal Care regulations, which conformed to the National Institutes
of Health Guidelines on the Care of Laboratory Animals.
Four adult Sardinian ewes, aged 5.5 years, weighing approximately 45 kg, were used
in the study. All animals were examined and found to be in good health.
Surgery was performed under sterile conditions and with sheep under general anesthesia.
All sheep were intubated after the administration of thiopentone (25 mg/kg) and ventilated
with O2 in N2O by volume control. Anesthesia was maintained with 1.5 to 2% of isoflurane; a bolus
dose of 0.1 mg of fentanyl was given before surgery.
A medial parapatellar arthrotomy was performed on the right and left stifle of all
animals. Incision was performed to expose the medial femoral condyle in both hind
legs. An 8-mm-diameter (area: 50.3 mm2), full-thickness chondral lesion in the load-bearing area of each medial femoral
condyle was created using an arthroscopic burr. The calcified cartilage layer was
removed; vertical walls were created at the periphery of the cartilage lesion. The
defects were then treated with microfracture on one side and nanofracture on the contralateral
side. Each cartilage lesion was treated with five bone channels. The distance between
each channel was 3 mm according to previous recommendations.[7] Microfracture sites were treated using an awl manufactured by a curved Steadman
awl. The perforation depth was user-controlled with visual feedback from the awl tip.
Nanofracture sites were treated using a cannulated awl and a 1-mm-thick Nitinol needle
(NanoFx, Arthrosurface Inc). The 9-mm perforation depth of the needle was controlled
by the awl ([Fig. 1]). The surgical technique was previously described.[9]
Fig. 1 (A) Treatment of the defect with microfracture. (B) Treatment of the defect with nanofracture.
Upon completion of the cartilage repair procedures, all incisions were closed in layers
according to standard surgical practice. Postoperatively, the animals were kept in
stalls with limited movement and weight-bearing. Animals were then left free to roam
in their fencings without any immobilization of the operated limb. Full weight-bearing
was allowed as tolerated, and no specific exercise regimen was adopted. General health
and weight-bearing status were monitored by a veterinary during recovery.
Immediately after euthanasia, the defects were photographed to allow assessment by
two blinded observers. Macroscopic evaluation was assessed based on the International
Cartilage Repair Society (ICRS) evaluation score ([Table 1]).
Table 1
International Cartilage Repair Society macroscopic evaluation of cartilage repair
|
Categories
|
Score
|
|
Degree of defect repair
|
|
|
In level with surrounding cartilage
|
4
|
|
75% repair of defect depth
|
3
|
|
50% repair of defect depth
|
2
|
|
25% repair of defect depth
|
1
|
|
0% repair of defect depth
|
0
|
|
Integration to border zone
|
|
|
Complete integration with surrounding cartilage
|
4
|
|
Demarcating border < 1 mm
|
3
|
|
¾ of graft integrated, ¼ with a notable border > 1 mm width
|
2
|
|
½ of graft integrated with surrounding cartilage, ½ with a notable border > 1 mm
|
1
|
|
From no contact to ¼ of graft integrated with surrounding cartilage
|
0
|
|
Macroscopic appearance
|
|
|
Intact smooth surface
|
4
|
|
Fibrillated surface
|
3
|
|
Small, scattered fissures or cracs
|
2
|
|
Several, small or few but large fissures
|
1
|
|
Total degeneration of grafted area
|
0
|
|
Overall repair assessment
|
|
|
Grade I: normal
|
12
|
|
Grade II: nearly normal
|
11–8
|
|
Grade III: abnormal
|
7–4
|
|
Grade IV: severely abnormal
|
3–1
|
Condylar articular defects containing regenerated tissue, adjacent host cartilage,
and subchondral bone were harvested using a water-cooled circular saw. The tissue
blocks were fixed in 10% neutral-buffered formalin for 4 days and then placed in a
decalcification solution for 4 to 10 days. After washing in running tap water for
4 to 8 hours to remove all traces of decalcification solution, the osteochondral specimens
were paraffin-embedded and 4-μ sections were stained with safranin orange/fast green
(Safranin O) and hematoxylin and eosin as previously described.[19]
For the immunohistochemical analysis, we used the avidin–biotin complex and peroxidase.
The immunohistochemical determination of type I and type II collagen contents was
conducted on paraffin-embedded sections using an automatic immunostainer (Ventana
BenchMark ULTRA, Roche Diagnostics, Basel, Switzerland), with a ready-to-use dilution
of a monoclonal mouse antitype I or antitype II collagen immunoglobulin (ThermoFisher
Scientific, Waltham, Massachusetts, United States). The reaction was displayed by
the Ultraview DAB kit (Ventana Medical System, Roche Diagnostics), a revelation system
kit including the secondary antimouse antibody biotinylated, the enzymatic substrate,
and the chromogen DAB.
Immunoreactivity to type I collagen in the repair tissue was compared with that of
the adjacent subchondral bone, serving as positive internal control (0 = no immunoreactivity;
1 = significantly weaker; 2 = moderately weaker; 3 = similar; 4 = stronger immunoreactivity).
Results
Macroscopic Evaluation
Macroscopic evaluations conducted according to the ICRS evaluation score on chondral
defects treated with microfracture technique showed a partial filling of the defect
by a thin healing tissue; the latter showed characteristics similar to the healthy
cartilage almost exclusively on the peripheral areas of the lesion ([Fig. 2]). Conversely, macroscopic evaluations performed on condyles treated by using the
nanofracture technique showed an almost complete covering of the defect by a newly
formed tissue, resulting more similar to the native cartilage in the whole chondral
defect. Furthermore, the repair tissue showed a higher integration with the surrounding
cartilage and lack of fibrillation and fissures on the majority of the surface ([Fig. 3]).
Fig. 2 Macroscopic appearance of a defect treated with microfracture technique 12 months
after surgery. Partial filling of the defect by a thin healing tissue can be observed.
Fig. 3 Macroscopic appearance of a defect treated with nanofracture technique 12 months
after surgery. The defect is almost completely covered by a newly formed tissue that
is similar to the native cartilage. Furthermore, the repair tissue shows a good integration
with the surrounding cartilage and lack of fibrillation and fissures on the majority
of the surface.
Histological and Immunohistochemical Evaluation
Histological evaluation on samples treated using the microfracture technique showed
a partial filling of the defect by fibrocartilaginous tissue formed by rounded cells
similar to chondrocytes submerged into a fibrous extracellular matrix. The repair
tissue did not show the normal structure consisting of cartilage layers and was characterized
by an almost complete lack of tidemark and a severe alteration of the subchondral
bone architecture ([Fig. 4]).
Fig. 4 Histological evaluation of a defect treated with microfracture technique 12 months
after surgery. Repair tissue does not show the normal structure consisting of cartilage
layers and is characterized by an almost complete lack of tidemark and a severe alteration
of the subchondral bone architecture.
On the other hand, the histological evaluation of samples treated with the nanofracture
technique highlighted a more satisfactory defect filling and a better structural cartilage
architecture into the lesion. The newly formed tissue showed the same features as
a fibrohyaline repair tissue in which small groups of rounded cellular elements clustered
and diffusely distributed could be observed ([Fig. 5]).
Fig. 5 Histological evaluation of a defect treated with nanofracture technique 12 months
after surgery. Satisfactory defect filling and restoration of the structural cartilage
architecture can be observed.
Immunofluorescence showed a scarce positivity for type II collagen in defects treated
with microfractures ([Fig. 6]) compared with defects treated by with nanofractures. In the latter group, the repair
tissue presented a strong positivity for type II collagen, visible especially around
the perforations ([Fig. 7]).
Fig. 6 Immunohistochemical evaluation of a defect treated with microfracture technique 12
months after surgery. The defect is filled with fibrocartilage tissue, with poor immunofluorescence
positivity for type II collagen.
Fig. 7 Immunohistochemical evaluation of a defect treated with nanofracture technique 12
months after surgery. (A) The defect is completely filled by fibrohyaline repair tissue strongly positive
to immunohistochemistry for type II collagen. (B) Strong positivity in correspondence of the channels can be observed. (C) Clones of regenerating chondrocytes are present around the area that correspond
to increased collagen type II synthesis.
Discussion
The main finding of this study is that small-diameter needle perforation into full-thickness
chondral defects significantly improves repair in a preclinical animal model at 12
months postoperatively compared with large drill holes.
Since the first use proposed by Steadman in the 1980s, microfracture has become the
treatment of choice for the management of chondral lesions.[3] The technique is based on direct stimulation of MSCs of the subchondral bone, which
have a high potential for differentiation into various connective tissues, including
cartilage, bone, tendon, and ligaments.[20]
[21] The regenerative effects of MSCs achieve their objective in promoting tissue repair
and the resolution of inflammation through direct cell-to-cell interaction or by secretion
of bioactive components.[22]
[23] Several studies confirmed the efficacy of the microfracture technique, with improved
clinical outcomes in 70 to 90% of patients.[7]
[8]
[13]
[24]
[25]
[26] However, the formation of fibrocartilage tissue and the deterioration of clinical
results over time represent important limitations,[27]
[28]
[29]
[30] and alternative procedures for the management of chondral lesions have been advocated.[14]
[15]
The use of standard microfracture awls is no longer supported by recent basic science
evidence.[17]
[31]
[32]
Eldracher et al conducted a study on an animal model aiming at evaluating through
macroscopic, histological, immunohistochemical, and microcomputed tomography analysis
the repair of chondral defects treated by a perforation diameter of either 1 or 1.8
m. Results were definitely better in defects treated with smaller diameter holes,
repair tissue having greater content in type II collagen and showing an architecture
more similar to normal.[33]
Orth et al recently published their comparative results treating 8 × 4-mm full-thickness
defects in the weight-bearing area of ovine knees. The study used two custom-made
awls with a diameter of either 1 or 1.2 mm. The perforation depth was stop controlled
at 5 mm; the distal tips were trihedral. The authors reported a significant improvement
in the overall histological score using small diameter awls. In addition, the histological
surface regularity was significantly improved by smaller instruments.[32]
Chen et al published several studies evaluating the histological characteristics of
the newly formed tissue after treatment of chondral defects with different reparative
techniques and various degrees of penetration into the subchondral bone.[17]
[31]
[34] Histological and histomorphometric evaluations showed that the best access to bone
marrow was observed when drilling to greater depths, thereby improving defect fill
and production of cartilage with a higher hyaline content.[25] A further study by the same group showed that treatment with microfracture is associated
with fragmentation and compaction around the trabecular canals.[17]
Our study confirmed previous results, showing beneficial subchondral effects using
small diameter needle perforation. While Orth et al[32] chose a 5-mm perforation depth using custom instruments, we used clinically available
instrumentation for nanofracture involving the use of a 1-mm-thick needle inserted
into a cannulated awl that creates stop-controlled 9-mm-deep perforations. This technique
was compared with microfracture using the standard conically shaped tip.
In accordance with different studies published in the literature, our work highlights
how chondral defects repair obtained with reduced diameter perforations and with a
higher penetration into the subchondral bone is associated with the formation of tissue
having histological and immunohistological characteristics more similar to the native
cartilage compared with standard microfracture. Better results obtained with nanofracture
might be explained by a lower fragmentation and trabecular compaction around the perforations,
leading to a better communication between those and the surrounding trabecular channels.
These canals allow for the immediate on-site formation of a superclot, resulting in
improved recruitment of multipotent MSCs.
Overall, inference of an animal study is limited because functional status cannot
be assessed at baseline and follow-up evaluations. However, the animal model used
in this study is considered suitable for cartilage defect testing. Limitations due
to small cohort size should also be considered.
Finally, no biomechanical analysis of the newly formed tissue was performed.
In conclusion, nanofracture represents a novel technique for bone marrow stimulation
based on smaller diameter and deeper perforations, resulting into a more satisfactory
chondral repair compared with methods based on perforation of larger diameter and
lower depth.
