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International Orthopaedics

, Volume 42, Issue 7, pp 1755–1767 | Cite as

Effects of press-fit biphasic (collagen and HA/βTCP) scaffold with cell-based therapy on cartilage and subchondral bone repair knee defect in rabbits

  • Jacques Hernigou
  • Pascale Vertongen
  • Esfandiar Chahidi
  • Theofylaktos Kyriakidis
  • Jean-Paul Dehoux
  • Magalie Crutzen
  • Sébastien Boutry
  • Lionel Larbanoix
  • Sarah Houben
  • Nathalie Gaspard
  • Dimitrios Koulalis
  • Joanne Rasschaert
Original Paper

Abstract

Introduction

Human spontaneous osteonecrosis of the knee (SPONK) is still challenging as the current treatments do not allow the production of hyaline cartilage tissue. The aim of the present study was to explore the therapeutic potential of cartilage regeneration using a new biphasic scaffold (type I collagen/hydroxyapatite) previously loaded or not with concentrated bone marrow cells.

Material and methods

Female rabbits were operated of one knee to create articular lesions of the trochlea (three holes of 4 × 4mm). The holes were left empty in the control group or were filled with the scaffold alone or the scaffold previously loaded with concentrated bone marrow cells. After two months, rabbits were sacrificed and the structure of the newly formed tissues were evaluated by macroscopic, MRI, and immunohistochemistry analyses.

Results

Macroscopic and MRI evaluation of the knees did not show differences between the three groups (p > 0.05). However, histological analysis demonstrated that a higher O’Driscoll score was obtained in the two groups treated with the scaffold, as compared to the control group (p < 0.05). The number of cells in treated area was higher in scaffold groups compared to the control group (p < 0.05). There was no difference for intensity of collagen type II between the groups (p > 0.05) but subchondral bone repair was significantly thicker in scaffold-treated groups than in the control group (1 mm for the control group vs 2.1 and 2.6 mm for scaffold groups). Furthermore, we observed that scaffolds previously loaded with concentrated bone marrow were more reabsorbed (p < 0.05).

Conclusion

The use of a biphasic scaffold previously loaded with concentrated bone marrow significantly improves cartilage lesion healing.

Keywords

Spontaneous osteonecrosis of the knee Cartilage repair Bone marrow cells Biphasic scaffold 

Introduction

Human spontaneous osteonecrosis [1] of the knee (SPONK) is a disorder characterized by a subchondral fracture of the medial condyle leading to collapse of the affected region associated with the development of necrotic tissue. Different stages were described (I to IV) regarding the severity of the disease evaluated by X-ray. Patients who progress to stage III or IV may receive surgical chondral defect repair. In this technique, subchondral bone is harvested with a piece of autologous articular cartilage from less weight-bearing surfaces and transplanted to the altered area. Surgical chondral defect repair is however restricted to patients presenting a localized and well-defined cartilage lesion in an attempt to avoid prosthetic implantation.

When autologous osteochondral tissue cannot be harvested from a healthy area and transplanted (mosaicplasty), other techniques may be used to ensure coverage of the affected area.

Another surgical option performed to treat localized cartilage defect is bone marrow stimulation; this technique, referred as the Pridie technique, is relatively simple, minimally invasive and cost-effective. The subchondral bone plate below the cartilage lesion is perforated to initiate bleeding and to induce a reparative cellular response. During this procedure, the perforated hole is kept as a non-treated empty defect for spontaneous regeneration. The principle behind this regenerative resurfacing strategy is the triggering of migration of non-differentiated bone marrow-derived multipotent mesenchymal stem cells (MSCs) from the subchondral bone into the defect site, leading to formation of new cartilage tissue [2, 3, 4]. Patients treated with bone marrow stimulation generally demonstrate clinical improvements up to 1.5 to three years after surgery. However, five years after surgery, higher incidence of clinical failure is observed [5, 6], most likely because the newly formed tissue generally consists of fibrocartilage tissue rather than hyaline cartilage. Fibrocartilage tissue incompletely fills the defect, integrates poorly with the surrounding tissue, and has inferior mechanical properties compared to hyaline cartilage due to the lack of arcade-like organization of the collagen fibers and lack of a well-defined zonal stratification of chondrocytes [7, 8].

To improve the mechanical properties of the regenerative tissue, different strategies have been applied including implantation of cell-free biomaterials (or scaffolds). This option offers various advantageous properties such as lack of donor-site morbidity and application of one-stage surgical procedures. Many researchers have investigated the use of various natural biomaterials in vivo, such as collagen, alginate, and hyaluronic acid (HA) or synthetic polymers as polycaprolactone, polyvinyl alcohol, and poly-lactic-co-glycolic acid [9]. To combine the advantageous properties of these materials, multilayered biomaterials have been constructed. Indeed, compositionally and morphologically graded scaffolds, made with the bio-hybrid composites, displayed the ability to support selective cell differentiation towards the osteogenic and chondrogenic lineages [10]. Moreover, the presence of scaffolds prevents fibroblast invasion of the graft that may otherwise lead to fibrocartilaginous tissue formation. Implantation of an osteochondral biomimetic scaffold consisting of a type I equine collagen cartilage-like layer combined to a mineralized layer of HA mixed with beta tri-calcium phosphate (βTCP) as subchondral bone compartment could be optimal to achieve hyaline cartilage and bone formation.

The aim of this experimental study was therefore to explore the potential of cartilage healing of a new biphasic scaffold composed of two layers: one of type I equine collagen and the second of HA/βTCP granules of 300 to 600 μm of diameter.

Engineered scaffold constructs, previously loaded or not with concentrated bone marrow mononuclear cells (BMMC), were implanted in osteochondral defects of rabbit knee joints. The two scaffold-treated groups were compared to a group of non-treated osteochondral defect.

The following questions were addressed: Is the use of an acellular biphasic scaffold an effective therapeutic option leading to production of hyaline cartilage and subchondral bone? Does cell therapy associated to this new scaffold improve cartilage and subchondral bone repair?

Material and methods

Animals

This study was carried out in strict compliance with ACUC international recommendations. Ethics approvals were granted by the Animal Ethics Committees.

Twenty-five female New Zealand white rabbits of 3.0 to 3.6 kg (CER Group, Marloie, Belgium—LA1800104) were randomly divided into three groups: a non-treated osteochondral defect group used as control (C group; 5 rabbits) and two experimental groups treated with the scaffold alone (SA group; 10 rabbits) or treated with the scaffold previously loaded with concentrated BMMC (SL group; 10 rabbits). Rabbits were operated in one knee to create three articular lesions of the trochlea (three holes of 4 mm; see below). Knee side was randomly selected. All rabbits were housed individually in cages. Then, eight weeks later, animals were sacrificed to evaluate cartilage and subchondral bone regeneration.

Surgical procedure

All surgeries were performed under aseptic conditions in a surgery room devoted to animal studies. Rabbits were anesthetized by subcutaneous injection of ketamine (30 mg/kg) and xylazine (5 mg/kg), and 1 ml lidocaine (5 mg/ml) was injected for local anesthesia. All rabbits received intramuscular buprenorphine (0.05 mg/kg) during surgery for analgesia.

Creation of empty osteochondral defects

A longitudinal cutaneous incision centered on the patella was performed and followed by an internal arthrotomy with external luxation of patella to expose the trochlea. Three holes of 4 mm of diameter and 4 mm of depth were performed with a wick of 4 mm and a slow speed drill to avoid surrounding bone burning (Fig. 1a). For the rabbits of the C group, the patella was reduced and the joint capsule, medial femoro-patellar ligament, and skin were closed.
Fig. 1

Pictures of the surgery procedure. a Repartition of the three holes in the trochlea. b Example of the three holes filled with patch of scaffold. c Injection of 50 μl of concentrated bone marrow in the scaffold

Thereafter, all rabbits were allowed to move freely without splints.

Implantation of acellular scaffold

For the 10 rabbits of the SA group, a 4-mm patch of scaffold was inserted in the three holes (Fig. 1b). All implanted scaffolds were cut into wafers with a diameter of 4 mm and were placed randomly into the knee defects. The granule layer was positioned towards the subchondral bone and the collagen layer was oriented towards the cartilage surface (Fig. 2a). The joint was closed as described previously.
Fig. 2

a Implantation of the scaffold with the collagen layer at the surface (axial view of the trochlea of a rabbit). b Macroscopic picture of the scaffold: the upper part collagen layer, lower part, and HA layer. c Scanning electron microscopy (SEM) view of the two layers of the scaffold

Scaffold with cell therapy

To isolate BMMC, percutaneous bone marrow aspiration was performed in the internal condyle of the contralateral distal femur of the unselected knee for surgery. The bone marrow sample (3 ml) was diluted with 1 ml of unfractionated heparin (1000 UI/ml) and phosphate-buffered saline (PBS) was added (vol/vol).

BMMC were then isolated by a modified method of Pittenger et al. [11]. Ficoll-Hypaque solution (4 ml) (1.077 g/cm3; Sigma, MO, USA) was put in sterile centrifuge tubes and 6 ml of bone marrow sample diluted in heparin and PBS were carefully layered over it. The samples were centrifuged at 300g for 20 minutes at 22 °C. After centrifugation, the plasma layer was carefully discarded and the BMMC (Fig. 3) were collected and transferred into another sterile tube. Sterile PBS was added to the cell suspension (final volume of 50 ml) and the samples were centrifuged for five minutes  at 500g at 22 °C. The supernatant was discarded and the BMMC pellet was suspended in PBS (final volume of 0.25 ml). BMMC were counted: the mean concentration of mononuclear cells was 21 × 106 cells/ml (IC95 10-32).
Fig. 3

Technique of concentration of bone marrow mononuclear cells using Ficoll®

Preparation of the scaffold previously loaded with concentrated BMMC

For the 10 rabbits of the SL group, 0.05 ml of autologous BMMC (on average 106 cells) were injected with a 25-G needle in each 4mm patch of scaffold before implantation in the different holes (Fig. 1b, c).

Scaffold

The biphasic and monolithic scaffold was developed by Novagenit (Mezzolombardo, Italy). It is composed of a layer of 4 mm of type I equine collagen in the upper part and a layer of 3 mm of HA/βTCP granules of 300 to 600 μm of diameter (Fig. 2b) in the bottom part forming a mineral-collagenic gradient similar to that of osteochondral connective. Because the thickness of cartilage is smaller in rabbits than in human, dimensions of the layers were reduced to 2 mm.

The internal structure of the HA layer is highly porous with the HA/βTCP granules being englobed into collagen (Fig. 2c). This prevents the granules after wetting from getting off the scaffold during the implantation procedure.

The surface of the collagen layer (chondral) is smooth with an underlying compact zone intended to avoid cell loss into the synovial lumen and thus hypertrophy.

Cell loading and subsequent short incubation demonstrate good bio-functional parameters in terms of fibroblast adhesion both on collagen and on granules.

Evaluation of cartilage and subchondral repair

Macroscopic and magnetic resonance imaging (MRI) evaluation

After sacrifice of animals, a “second look” was performed by the same approach to visually evaluate cartilage regeneration using the ICRS cartilage repair assessment score [12]. This score which was developed for human had to be adapted to the dimensions of the knee of rabbit for the item “integration border zone” (Table 1).
Table 1

ICRS cartilage repair assessment score and MRI MOCART score

ICRS cartilage repair assessment score

Points

MRI MOCART score

Points

Degree of defect repair

 

Degree of defect repair

 

 In level with surrounding cartilage

4

 Complete

20

 75% repair of defect depth

3

 Hypertrophy

15

 50% repair of defect depth

2

 Incomplete > 50%

10

 25% repair of defect depth

1

 Incomplete < 50%

5

 0% repair of defect depth

0

 Subchondral bone exposed

0

  

Integration border

 

Integration to border zone

 

 Complete

15

 Complete integration

4

 Demarcating border

10

 Demarcating border on 1/4

3

 Defect < 50% perimeter

5

 Demarcating border on 1/2

2

 Defect > 50% perimeter

0

 Demarcating border on 3/4

1

Surface of repair tissue

 

 No contact

0

 Intact

10

  

 Damaged < 50% of depth

5

Macroscopic appearance

 

 Damaged > 50% of depth

0

 Intact smooth surface

4

Structure

 

 Fibrillated surface

3

 Homogeneous

5

 Small, scattered fissures or cracks

2

 Heterogeneous

0

 Several, small or few but large fissures

1

Signal

 

 Total degeneration of grafted area

0

 Isointense

30

 

 Moderately hyper-/hypo-intense

10

 Markedly hyper-/hypo-intense

0

Subchondral bone

 

 Intact

5

 Disrupted

0

Subchondral lamina

 

 Intact

5

 Damaged

0

Effusion

 

 Yes

5

 No

0

Adhesion

 

 Yes

5

 No

0

Rabbit legs and the entire knees were dissected and conserved in PBS (4 °C) for MRI and histological examination. Three animals of the control group and six of the SA and SL groups were randomly selected and MRI was performed (after fixation of knees; see below) to evaluate cartilage regeneration using the MOCART score [13] (Table 1). Three test knees were studied before this work to ensure that there was no impact of the fixation on the MRI signals of the different tissues. Rabbit legs were roughly dried before being placed in a plastic bag and positioned in the MRI coil. MRI was performed at room temperature on a Bruker Biospec (Ettlingen, Germany) equipped with a 9.4-T magnet, using a volume coil of 40 mm diameter allowing for a field of view (FOV) of 5 cm length (scheduled for mouse body). 3D acquisition sequences were set up using the Paravision 5.1 software (Bruker, Ettlingen, Germany), with a resolution of 0.195 mm × 0.195 mm × 0.195 mm (FOV of 2.5 cm × 2.5 cm × 5 cm). Gradient echo FISP (fast imaging with steady-state precession) T1-weighted images (repetition time (TR), 5.3 ms; echo time (TE), 1.6 ms; flip angle, 15° or 30 °; NEX, 4, 12 min 40 s 778 ms of acquisition time) were collected. Legs were quickly replaced in their PBS container after the MRI session.

Histological examination

Knees were fixed by paraformaldehyde (4%—Yvsolab, Turnhout, Belgium) during 3 days and then preserved in PBS at 4 °C. At this stage,15 knees underwent MRI (see above). After which, they were decalcified using ethylene-diamine-tetra-acetic acid (EDTA; Avantor, Netherlands) for 28 days (concentration, 12.5%; pH 7.2) and embedded in paraffin after being washed. Slices of 4 μm thick were performed and disposed on Superfrost Plus™ (Thermo Scientific, MA, USA) slides for every hole performed during surgery.

Immunohistochemistry (IHC) protocols for rabbit collagen type II staining

The immunohistochemical labelling was performed using the ABC method. Briefly, slides were warmed at 60 °C during 30 minutes and were then dewaxed by toluene (two baths) and alcohol 100% (two baths). After a 30 minute treatment with H2O2 (0.3%) in methanol to inhibit endogenous peroxidase, slides were washed five minutes in Tris buffer saline (TBS; 0.01 mol/L Tris, 0.15 mol/L NaCl, pH 7.4) and incubated at 37 °C in trypsin (0.1% in TBS) during 30 min and after three washings (10 min each) in TBS, the slides were incubated with the blocking solution (10%) (v/v) normal horse serum in TBS.

After overnight incubation at room temperature with the primary antibody (Anti-Human Collagen Type II Mouse Monoclonal Antibody [clone: II-4C11], MP Biomedicals, CA, USA), diluted 1/250 in TBS containing 1% normal horse serum, the slides were washed in TBS (3 × 5 minutes) and sequentially incubated with secondary antibody conjugated to biotin (diluted 1/100 in TBS + normal horse serum 1%). Slides were washed again (TBS 3 × 5 minutes) and the signal amplified by the ABC kit (Vector, CA, USA) for 30 minutes and washed (TBS 3 × 5 minutes) The peroxidase activity was revealed using diaminobenzidine (DACO) as chromogen and stopped with distilled water [14]. The slides were counterstained with haematoxylin [15] before dehydration in croissant baths of alcohol and two baths of toluene. They were mounted in D.P.X. (Sigma-Aldrich, MO, USA).

To quantify collagen type II labelling, we measured the stained surface (in brown) and the intensity of the color for both normal and newly formed tissue of the same slide using the ImageJ software (MD, USA). Then, intensity of newly formed cartilage was expressed in percentage of the intensity of normal cartilage, considered as 100%.

Slide observation and scanning

Tissue sections were examined with an Axioplan I microscope (Carl Zeiss, Oberkochen, Germany). Then, slides were scan at a 20× magnification and examined using Gateway Slide Path (Leica Biosystems, Nussloch, Germany) and ImageJ (MD, USA) free softwares after calibration of pixel size.

O’Driscoll score

For each slide, the modified O’Driscoll score was calculated [16, 17]. Mean total scores were compared between each group. For item 1 (cell morphology), when there was a heterogeneous tissue, the most important tissue was considered for scoring.

The thickness of cartilage was measured on each slide at three different positions of the newly formed cartilage and of both sides of the normal adjacent cartilage. Then, we compared the new cartilage with the normal adjacent cartilage on each slide. The maximum total score was 23 points.

Subchondral bone additional analysis

The higher thickness of subchondral bone was also measured using the ImageJ software. The number of HA granules were counted in the SA and SL groups and compared.

Cell count

Cells were counted using ImageJ (MD, USA) counter cells plugin on three positions of each slide: the center and each extremity. The counting surface was also measured and selected as one third of each new cartilage area. The mean concentration of cells/mm2 per slide in each group was calculated and compared.

Statistics

The results of macroscopic (ICRS and MOCART) and microscopic analyses (O’Driscoll score, cell count, subchondral bone analysis, and IHC) were graded by two independent blinded orthopaedic surgeons (the group of each animal was unknown). The Xlstat® software (Addinsoft, Paris, France) was used for statistics analysis. ANOVA statistical test followed by bilateral Dunnett’s test was used. For the comparison of the number of HA granules in SL and SA groups, we used Student’s t test. When percentage has been compared, we used a chi2 test. The results were considered as significant when p was < 0.05.

Results

All rabbits survived the surgery and during the two post-operative months of follow-up. Four rabbits presented wound dehiscence. They were treated by a quick re-intervention (subcutaneous lavage and new cutaneous suture) and prolonged antibiotics. No rabbit presented uncontrollable pain leading to the sacrifice of the animal.

Pre-histological examination of the three holes performed in the trochlea showed that the most proximal hole was not located in a cartilage area. It was therefore excluded from the study. Fifty holes were thus usable for histologic evaluation and slides of these holes were performed. The flow chart is resumed in Fig. 4.
Fig. 4

Flow chart of the study. C, control; SA, scaffold alone; SL, scaffold loaded with BMMC

Macroscopic findings

After animal sacrifice and joint opening, we did not observe significant changes of the synovia. The synovial tissue had a normal appearance without signs of inflammation. The synovial fluid was clear and seemed to have a normal viscosity and consistency (optical and tactile evaluation). Macroscopically, the host cartilage surrounding the defects showed a usual transparent white/bluish color.

In the control group, the defect area was clearly visible at the time of surgery. At the second look, the defect area was fully filled with repair tissue in all knees examined. The repair tissue was white and was nearly completely present at the edge of the defect (Fig. 5a (I)).
Fig. 5

Examples of macroscopic (I) and MRI (II) aspects of the central hole of each group. a Control group. b Scaffold alone group. c Scaffold loaded with BMMC group. Column I: macroscopic aspects of the holes; the blue arrows point the three fully filled holes. Column II: axial T1-weighted MRI view of the hole. The cartilage appears in hyper-signal (white dashed arrow) and subchondral bone appears in hypo-signal (white arrow)

In the SA and SL groups, the defect area was also totally filled with repair tissue in all knees at eight weeks follow-up. The repair tissue consisted of a mixture of white hard and translucent soft tissue. It was present at both the edge and the bottom of the defect, showing a close to normal repair level (Fig. 5b (I), c (I)). In line with these observations, the ICRS score on 12 points, adapted to the size of rabbits (Table 1), showed no significant differences (p > 0.05) between the three groups with a mean of 10.1 points for the control group, 10.4 points for the SA group, and 10.5 points for the SL group (Table 2).
Table 2

Macroscopic evaluations according to ICRS cartilage repair assessment score and to MRI MOCART score

 

Control group (IC95)

SA group (IC95)

SL group (IC95)

p ANOVA

ICRS cartilage repair assessment score

 Defect repair

3.8

3.7

3.3

 

 Border zone

3.8

3.3

3.6

 

 Macroscopic appearance

2.6

3.4

3.5

 

 Total

10.7 (9.3–10.8)

10.5 (10–11)

10.5 (10–10.9)

p > 0.05

MRI MOCART score

 Degree defect repair

20

19

16.7

 

 Border

15

15

15

 

 Surface

6.7

8

5

 

 Adhesion

5

5

5

 

 Structure

3.3

2

4.2

 

 Signal

30

30

30

 

 Effusion

5

5

5

 

 Subchondral lamina

5

5

5

 

 Subchondral bone

5

5

5

 

 Total/100

95 (94–96)

94 (91–97)

91 (87–95)

p > 0.05

MRI evaluation performed on 15 rabbits (3C, 6 SA, and 6 SL), 8 weeks after surgery, showed that the defect areas were entirely filled, confirming the visual observations. The cartilage presented a comparable thickness and a similar signal than the host cartilage located around the defects for each rabbit. A total peripheral integration of the new cartilage was observed in all knees, without effusion. Moreover, for all holes examined, a complete adhesion of the newly formed cartilage to the subchondral bone was observed.

For subchondral bone, T1-weighted MRI showed hypo-signal under the cartilage in the knees of SA and SL groups (Fig. 5b (II), c (II)). This was the consequence of an increased thickness and density of subchondral bone in these groups (cf subchondral results).

There was no significant difference between the three groups for the MOCART score (on 100 points), with 95, 94, and 91 points for the control, SA, and SL groups respectively (p > 0.05). Results are reported in Table 2.

Histological findings

From a histological point of view, all defects were fully filled with tissues. However, IHC slices presented heterogeneity regarding tissue organization in both the control and the two experimental groups. Indeed, in each group, some slices presented a homogenous well-organized tissue structure, with total or partial differentiation in hyaline cartilage (Fig. 6a, b) while others showed heterogeneous tissue associating fibrocartilage and partially differentiated hyaline cartilage (Fig. 6c).
Fig. 6

Presentation of multiples homogeneity of newly formed cartilage tissue. a Homogeneous tissue with newly formed tissue presenting a cartilage architecture. Rated with 4 points for “tissue morphology” item of the O’Driscoll score. b Homogeneous tissue with newly formed tissue partially differentiated in hyaline cartilage. Rated with 2 points for “tissue morphology” item of the O’Driscoll score. c Heterogeneous tissue with partially differentiated tissue in hyaline cartilage on the periphery and fibrocartilage at the center. Rated with 0 points for “tissue morphology” item of the O’Driscoll score. d Enlargement of the black square of a. We can observe arcade-like organization of the collagen type II fibers and a well-defined zonal stratification of chondrocytes, with still some clustering of chondrocytes. e Enlargement of the black square of b. Compared to d, chondrocytes and collagen type II fibers are less organized. f Enlargement of the black square of c. Association of a partially differentiated hyaline cartilage (blue nucleus and brown coloration) and fibrocartilage presenting no collagen type II fibers (blue nucleus and no brown coloration) and no organization of chondrocytes

The total O’Driscoll score was statistically higher (p < 0.05) in SA and SL groups compared to the control group, with 17.6 points for the SA and SL group versus 16.4 points for the control group. This was mainly due to a better integration of the new cartilage with the host cartilage around defect sites (Fig. 7) and to an enhanced restitution of subchondral bone (as described below) in the two scaffold-treated groups (Table 3).
Fig. 7

Presentation of different border integration results. a, b Two examples of no integration of the new cartilage with normal adjacent cartilage (black arrows), from the same slide of the control group. Rated with 0 points for “border integration” item of the O’Driscoll score. c, d Two examples of integrated new cartilage (black arrows), from the same slide of SA group. Rated with 2 points for “border integration” item of the O’Driscoll score

Table 3

Histologic O’Driscoll score

 

Control group (IC95)

SA group (IC95)

SI group (IC95)

p ANOVA

Tissue morphology

1.78

1.45

1.71

 

Integrity of surface

2.33

1.83

1.79

 

Thickness

2.00

2.00

1.86

 

Hypo-cellularity

3.00

3.00

3.00

 

Chondrocyte clustering

1.67

1.59

1.43

 

Degenerative changes

3.00

3.00

3.00

 

Subchondral bone restoration

1.72

3.03

3.07

 

Border integration

0.89

1.72

1.79

 

Total

16.39 (15.6–17.2)

17.62* (17–18.3)

17.64** (17–18.3)

p = 0.033

*p = 0.035 SA compared to the control group, ANOVA followed by bilateral Dunnett’s test;**p = 0.033 SL compared to the control group, ANOVA followed by bilateral Dunnett’s test

The thickness of the new cartilage was measured at three different positions of each slide and compared to the thickness of host cartilage around defect size. There was no difference between the groups (p > 0.05).

When examining the tissue morphology to evaluate the O’Driscoll score, we noticed that slides presented heterogeneous tissues (Fig. 6). When the center of the slide was composed of a hyaline cartilage tissue or a partially differentiated hyaline cartilage, borders were always composed of a similar or a more developed tissue. It was not observed for the opposite configuration. In scaffold groups (SA and SL), extremities were composed of a tissue of hyaline cartilage or a tissue partially differentiated in hyaline cartilage in 61% slides, whereas only 37% of the slides showed a differentiated tissue at the center (p < 0.0001; chi2 test).

The O’Driscoll score does not take in consideration the exact number of cells and the amount of collagen type II present in the newly formed cartilage.

Despite the fact that we did not observe hypo-cellularity in the different groups, the number of cells in the new cartilage area was different between the three groups. It was increased by 35 and 23% in the SL and the SA groups, respectively. These results were statistically different to control group only for SL group (p < 0.05). Cells were counted on one third of the entire new cartilage surface divided in three areas (each border and center of the new cartilage).

For tissue morphology, the O’Driscoll score is limited to a visual evaluation. It does not quantitatively take in consideration the composition of extra cellular scaffold. However, intensity and surface of collagen type II staining were not different in the three groups (p > 0.05; Table 4).
Table 4

Histological and immunohistochemistry aspect of new cartilage and subchondral bone in knees defects

 

Control group (IC95)

SA group (IC95)

SI group (IC95)

P ANOVA

Mean value of cartilage thickness (μm) and comparison to normal adjacent cartilage (%)

431 (377–485)

− 5%

409 (360–459)

0%

484 (402–566)

+ 1%

p > 0.05

Cells count in new cartilage

 Number of mononuclear cells/mm2

1443 (1091–1758)

1778 (1548–1929)

1943* (1721–2122)

p = 0.04

 Surface analyze for each slide mm2

0.7

0.7

0.9

Immunohistochemistry targeted on collagen type II

 Marked area mm2

0.84 (0.22–1.45)

0.68 (0.37–1)

1.22 (0.53–1.92)

p > 0.05

 Intensity of collagen type II labelling

95.8% (86.7–104.9)

96.1% (89.6–102.7)

95.1% (86.7–103.5)

Subchondral bone

 Mean thickness (mm)

1.05 (0.83–1.28)

2.63* (2.36–2.9)

2.12* (1.65–2.69)

p < 0.0001

 Number of HA

20** (15.9–24.7)

9** (5.8–12)

*p < 0.05 SA and SL compared to the control group, ANOVA followed by bilateral Dunnett’s test; **p < 0.0005 SA group vs SI group, Students’ t test

Thickness of subchondral bone and number of HA were evaluated (Fig. 8). The subchondral bone was twice thicker in both SA and SL groups compared to the control group (Table 4). The number of HA granules was reduced twice in the SL group when compared to the SA group (p < 0.0005). As shown on Fig. 8a the C group presented a bad restoration of subchondral bone when compared to subchondral bone restoration of the scaffold group (Fig. 8b, c).
Fig. 8

Presentation of subchondral bone restitution in the different groups. a Control group presenting multiple interruptions between newly formed cartilage and subchondral bone (black arrow). b SA group with many HA granules (> 22; dashed arrows) and no interruptions between newly formed cartilage and subchondral bone (black arrow). c SL group showing 7 HA granules (dashed arrows) and no interruptions between newly formed cartilage and subchondral bone (black arrow)

Discussion

Cartilage lesions may be caused by acute trauma, osteoarthritis, or osteonecrosis. If cartilage lesions are restricted to the articular cartilage, they are termed chondral or partial thickness defects and if the lesions penetrate into subchondral bone, they are called osteochondral or full thickness defects.

It is well-known that articular cartilage repair represents a therapeutic challenge due to the low capacity of cartilage for self-healing [18, 19, 20, 21]. With the lack of blood supply, complex biochemical events that usually take place in order to repair tissue damage cannot occur. Wound repair in hyaline cartilage is further prevented by the high density of the extracellular matrix of cartilage impairing the migration capacity of chondrocytes. As consequence, no repair process usually occurs in defects limited to the chondral part. On the contrary in osteochondral defects, a repair process is usually initiated by undifferentiated mesenchymal stem cells (MSCs) from the bone marrow tissue of subchondral bone. However, this repair is usually limited, depending on the patient age, cause of lesion (trauma, osteoarthritis, inflammatory disease), defect size, and location. Small full thickness defects caused by trauma in children have been sometimes observed to be repaired by formation of hyaline cartilage; in contrast, large osteochondral defects are only repaired by formation of scar tissue (fibrous tissue) or fibrocartilage. Therefore, in the past, the regenerative treatment option for joint cartilage particularly in the knee was identified as marrow stimulation techniques, including microfracture, Pridie drilling, and abrasion arthroplasty, all of which involved drilling holes through the subchondral plate to stimulate hyaline cartilage or at least fibrocartilage repair tissue. However, fibrocartilage is a less organized tissue compared to hyaline cartilage, containing mostly collagen type I with inferior mechanical and biochemical characteristics compared to normal hyaline articular cartilage, leading to failure of fibrocartilage matrix that breaks down and with time development of a secondary osteoarthritis.

The present study was therefore designed as a preliminary investigation to determine the feasibility of using a newly designed biphasic scaffold previously loaded with autologous bone marrow concentrate as a “single-step repair method” for cartilage surface and subchondral bone reconstruction. Out of 20 rabbits used, none showed a negative effect of biomaterial implantation on cartilage and subchondral bone regeneration. Our results demonstrated the in vivo efficiency of the new biphasic scaffold to repair osteochondral defects in rabbits. The repair process of subchondral bone was significantly increased when the defect was filled by the scaffold as compared with empty defects; furthermore, the number of cells in the new cartilage area was increased by 35% when the matrix was previously loaded with concentrated bone marrow. Lastly, the presence of the scaffold improved integration of the newly formed cartilage with the host cartilage around the defect sites.

This scaffold developed by Novagenit is composed of a layer of 4 mm of type I equine collagen and a layer of 3 mm of HA/βTCP granules englobed into collagen to assure porosity. For the superficial layer, collagenous matrix was chose to guide the infiltration, proliferation, and differentiation of bone marrow stem cells as proposed by Buma [4]. The cross-linked type I collagen was preferred as compared to type II collagen since in previous studies, type I collagen scaffolds were more efficient for cartilage repair [4]. Equine collagen was preferred as compared to bovine or porcine collagen since it has been used in dermatology as well as in plastic and vascular surgery for wound healing and skin ulcer therapy for at least three decades [22]. This equine collagen has no allergic reaction reported in more than three decades of use, showing a high degree of safety. The deeper layer with granules of HA/βTCP displaying a highly structured surface was considered as a method to mimic a bone structure and provide a rapid and efficient osteogenesis activity in the bone. Our study validates such a proposal with thick subchondral bone in scaffolds treated animals as compared to the bad restoration of subchondral bone in empty defects. This metabolic high regeneration of bone is different of the slower chondrogenic regeneration. Therefore, the collagen surface in contact with HA may have the advantage to act as a barrier to inhibit angiogenesis and excessive osseous growth which would not be suitable into the chondral layer. This was observed in our study as the histological data showed that the interface between hyaline-like cartilage and bone-like tissue was situated at the site of the junction between the two layers of the matrix.

We loaded our scaffold with autologous bone marrow concentrate before implantation. One advantage of using bone marrow rather than chondrocytes or MSC cultures is that it allows a single-step repair method, avoiding cell manipulation and two consecutive surgical procedures. To perform a “1-step cartilage repair process,” improving the cell source is the first problem to be solved. In the past, this was done by the microfracture technique. Microfracture induces MSCs and chondroprogenitors cells migration and enhances cytokine release to promote cartilage repair [5, 6]. This method commonly used in clinics, however, usually leads to a fibrocartilage formation rather than a hyaline cartilage [5, 6]. The reason is probably the recruitment of a limited number of stem cells from the bone marrow. Since the numbers of MSCs in BM aspirates are low, we concentrated our bone marrow aspirate as usually performed in clinical practice. As a consequence, our results revealed a large number of cells in cartilage of the rabbits that had received scaffold previously loaded with BMMC. The higher cell regeneration of osteochondral tissue in the rabbits treated with bone marrow concentrate could be attributed to the presence and the differentiation of MSCs supplied by the bone marrow concentrate. Although the bone marrow cells had not been labelled in our experiment, the histological results showed that a lower number of host cells penetrated into the constructs in the cell-free group, supporting the hypothesis that the cells within the defect originated from the bone marrow concentrate. MSCs could also have derived from the synovial membrane towards the surface of the collagen [23].

Our results suggest that cartilage healing from the scaffold has a centripetal evolution with better results in the periphery than in the center of the defect (better border integration and better tissue differentiation in hyaline cartilage). This might be due to the contact with normal adjacent cartilage. These findings have been described by Clavé et al. in his work in 2016 comparing a patch supporting autologous chondrocytes to mosaicplasty [24]. They observed the opposite in for mosaicplasty with a less good peripheral integration. Based on these findings, the combination of a biphasic scaffold with a graft of mosaicplasty at the centre could be an interesting tool to suppress inconvenient of each technic for large lesions.

We choose to include only healthy animals receiving biomaterials with a normal cartilage outside the area of the defect as it is observed in osteonecrosis. As in osteonecrosis, there is a poor vascularization [25], and the results might not be as good as in our experiment. However, it has been demonstrated that cell migration is possible in type I collagenous matrix without increased vascularization [4] implying that this technique could be used in an osteonecrosis model in clinical practice.

The present study has some limitations: (1) Wei and Messner [26, 27] found that spontaneous healing of osteochondral defects in the knee joints of non-skeletal mature rabbits resulted in a faster filling and earlier tissue specialization than in adolescent or older skeletal mature animals. Even though our rabbits were skeletally mature, their relative young age could have had some beneficial effect on the self-repair capacity in the control group. Indeed, we observed growth plate in some animals testifying their relative young age. (2) A limitation of comparison with other studies is that the specific topics addressed in the scoring systems greatly differ: some reports focus only on the regeneration of cartilage; others on cartilage as well as subchondral bone; some include a biomaterial component (e.g., scoring degradation of the implant). (3) The anatomical and biomechanical cartilage characteristics of rabbit knees are different from human knees. The cartilage repair in a rabbit model has therefore some limitations, as for example, the thinner hyaline cartilage layer in rabbits as compared to humans. Nevertheless, the biological aspects of cartilage in rabbits are similar to those of humans [28].

Conclusion

The original biphasic scaffold used in our study to repair articular osteochondral defect in a one step cell-based approach allowed the production of hyaline cartilage in rabbits. The implantation of the bilayer scaffold alone demonstrated less resorption of the implant. The preparation of bone marrow concentrated, as used in clinical practice, appears to favor cell differentiation into bone-like and cartilage-like tissue in vivo, according to the different layer composition. Further study is needed to confirm whether this regenerated tissue would not degenerate after a long period. This investigation was performed following the protocol of a real clinical procedure and suggests that non-cultured MSCs from bone marrow aspirate can proliferate on this scaffold. Our data support therefore clinical trials with the biphasic scaffold for orthopaedic clinical applications.

Notes

Funding

This work was supported by a research subvention of Novagenit Srl and a research grant of the “Société Royale Belge de Chirurgie Orthopédique et de Traumatologie” (SORBCOT).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and institutional guidelines for the care and use of animals were followed. All procedures performed in the study were in accordance with the ethical standards of the institution.

Ethics approval was granted by Université libre de Bruxelles (ULB) Animal Ethics Committee (618N June 2016) and Université catholique de Louvain (UCL) Animal Ethics Committee (2016/UCL/MD/014 August 2016).

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Copyright information

© SICOT aisbl 2018

Authors and Affiliations

  • Jacques Hernigou
    • 1
    • 2
  • Pascale Vertongen
    • 2
  • Esfandiar Chahidi
    • 1
  • Theofylaktos Kyriakidis
    • 3
  • Jean-Paul Dehoux
    • 4
  • Magalie Crutzen
    • 4
  • Sébastien Boutry
    • 5
  • Lionel Larbanoix
    • 5
  • Sarah Houben
    • 6
  • Nathalie Gaspard
    • 2
  • Dimitrios Koulalis
    • 3
  • Joanne Rasschaert
    • 2
  1. 1.Department of Orthopaedic and Traumatology SurgeryEpiCURA HospitalHornuBelgium
  2. 2.Laboratory of Bone and Metabolic Biochemistry, Faculty of MedicineUniversité libre de BruxellesBrusselsBelgium
  3. 3.Department of Orthopaedic and Traumatology Surgery – Erasme HospitalUniversité libre de BruxellesBrusselsBelgium
  4. 4.Institute of Experimental and Clinical Research (IREC), Laboratory of Experimental Surgery and Transplantation (CHEX)Université catholique de LouvainBrusselsBelgium
  5. 5.Center for Microscopy and Molecular Imaging (CMMI)Université de Mons (UMONS)CharleroiBelgium
  6. 6.Laboratory of Histology, Neuroanatomy and Neuropathology, Faculty of MedicineUniversité libre de BruxellesBrusselsBelgium

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