A comparative study of tissue-engineered constructs from Acropora and Porites coral in a large animal bone defect model

Animals

A total of 15 healthy, two-year-old, Pré-Alpes sheep (60 kg) were obtained from a licensed vendor (INRA, Jouy-en-Josas, France) and raised in accordance with European laws (Directive 24.11.1986.86/609/CEE). Animal housing and care were carried out using procedures described in previous publications by members of our team.^6,7 All procedures were performed in compliance with legislation concerning animal experimentation and approved by the Ethical Committee.

TEC preparation

Autologous MSCs were isolated from bone marrow harvested from the sheep iliac crest and amplified as previously described until the second passage (supplementary material 1).⁸ Coral cubes (3x3x3 mm³) from either Acropora or Porites (Biocoral France, Saint-Gonnery, France) were used as scaffolds. Acropora coral has heterogeneously dispatched and interconnected large pores (412 μm, standard deviation sd 212 μm) and high permeability (4.46x10^-9 m²), whereas Porites coral has homogeneously dispatched and interconnected smaller pores (154 sd 53 μm) and low permeability (0.12x10^-9 m²).¹¹ For each genus, the cube specimens (n = 10) were imaged with high-resolution microCT (80 kV source voltage, 100 mA source current, 7.91 μm pixel size, 180° rotation, 0.3 second exposition), and reconstructed and analysed to determine their macroscopic architectures using dedicated software (Skyscan 1172, NRecon and CTAn; Skyscan, Aartselaar, Belgium). All cubes were sterilised by autoclaving (at 121°C for 20 minutes), a method that is known to preserve coral composition and structure.^11,15 Sterile coral cubes were washed with phosphate-buffered saline (PBS), immersed in culture medium for 24 hours, and subsequently loaded into a custom-made perfusion bioreactor containing culture medium, such as α-Minimum Essential Medium Eagle (Sigma-Aldrich, St Louis, Missouri) -10 %Fetal Bovine Serum (Sigma-Aldrich) at 10⁵ cells/cube, as previously described.¹⁶ The bioreactor was operated under sterile conditions at 10 ml/min flow at 37°C for seven consecutive days. The medium was changed every three days.

In order to control the distribution and presence of living MSCs onto TECs, three cubes from each group were randomly chosen on the day of surgery and the MSCs were labelled using carboxyfluorescein diacetate succinimidyl ester (CFSE) according to standard techniques. CFSE covalently labelled long-lived intracellular molecules with a fluorescent dye. Thus, the dye, examined under fluorescence microscopy, revealed living cells onto the cubes.¹⁶

Surgical procedures

Sheep were randomly assigned into three groups according to whether the CSD was filled with Acropora-TEC (n = 7), Porites-TEC (n = 6) or fragmented corticocancellous autograft harvested from the iliac crest (n = 2). In respect to the principles of the three Rs, as defined by Russell and Burch¹⁷ the number of animals in the positive control group was reduced because previous experiments, by our team, have shown this model to consistently result in bone union in the same model in sheep.^6,7,9,14 The surgical procedures (supplementary material 2) were performed under general anaesthesia in aseptic conditions as previously described and validated.¹⁴ In brief, a 25-mm long mid-diaphyseal ostectomy was performed in the metatarsal bone with full periosteal elevation. The so-created large defect was stabilised by plate (3.5 Dynamic Compression Plate, Synthes, Etupes, France) and the resected bone was fully replaced with TECs or autograft. Craniocaudal radiographs of the operated limb were obtained at the end of surgery, and at two and four months after surgery. The animals were killed after four months, using a barbiturate overdose.

Specimen collection and analysis

Immediately after killing, all the treated metatarsal bones were excised and fixed in 10% neutral buffered formalin for two weeks. The stabilisation plates were then removed and the implant sites, along with 2 cm of the surrounding host bone on each edge were removed. These pieces were embedded in methyl methacrylate resin according to established techniques⁶ and kept at room temperature until sectioning.

MicroCT scan analysis

All embedded specimens were imaged and analysed with a high-resolution microCT (Skyscan1172; Skyscan) with 80 kV source voltage, 100 mA source current, 26.6 μm pixel size, 180° rotation, 0.2 seconds exposition time, frame averaging 20, and aluminium-copper filters. On average, 1260 slides per sample were reconstructed using NRecon software (Skyscan). Data were treated using a global fixed threshold (60 to 220 grey levels) with the same volume of interest, corresponding to a cylinder centred in the middle of the defect with a length equal to the longest defect.

For qualitative analysis of bone formation, lateral, medial, cranial and caudal cortices were examined, and the number of united cortices was recorded for each specimen. We considered two parameters for the qualitative evaluation of the bone formation: (i) if at least one of the four cortices were united by a bony bridge, we called it “bone union”, and (ii) if all the four cortices presented union, we considered that “bone regeneration” was achieved.

The diameters of both the proximal and distal parts of the metatarsal bone and the volume of the resected bone were measured to access group comparability.

For quantitative volume analysis of the new bone and for the residual coral within the region of interest (ROI), dedicated software (CTAn; Skyscan) was used to obtain the bone volume (BV) with bone-specific threshold (60 to 140 grey levels) and the coral volume (CoV) with coral-specific threshold (140 to 220 grey levels). The scaffold bioresorption rate was measured and expressed as a percentage of the initial coral volume ICoV (125 coral cubes imaged by microCT without being implanted), as followed: (ICoV-CoV) / ICoV. Subsequently, the ROI region was divided into three equal parts corresponding to the proximal, central and distal areas. The same microCT analyses used for BV and CoV were performed.

Concerning the autograft group, the volume of newly formed bone could not be distinguished from the implanted bone, thus the overall bone volume was assessed, but statistical comparison could not be performed.

Undecalcified histology

All embedded metatarsal bone specimens were cut lengthwise using a circular saw (200 to 300μm, Leitz 1600; Leica Biosystems, Nussloch, Germany). The section closest to the longitudinal mid-sagittal plane was selected for histological analysis, ground down to 100 μm thick, polished and stained. The staining protocol included successive baths: Stevenel blue bath at 60°C during 15 seconds, water, van Gieson Picrofuchsin during 60 seconds and 100% alcohol. It permitted to discriminate by staining bone matrix, cell nuclei, and coral scaffold (in red, blue and brown, respectively).

Statistics

Statistical analyses were performed using a commercially available software package (GraphPad Prism V6.0c; GraphPad Software Inc., La Jolla, California). Quantitative data were expressed as mean and sd and analysed using the t-test or the Mann-Whitney test depending on the results of the normality test. The Kruskal–Wallis test was performed for comparison between more than two groups. Linear regression was used to determine correlations between quantitative data; the regression coefficient was stated as R². The significance level was set at p < 0.05.

In vitro evaluation

The two scaffolds exhibited different microCT architecture (Fig. 1 and Table I): Acropora displayed large and irregular pores with heterogeneous distribution, whereas Porites had more homogeneously distributed smaller pores; Acropora porosity was also lower than that of Porites. For the two types of coral genera tested, fluorescent microscopy observations of scaffolds loaded with CFSE-labelled MSCs revealed the presence of adherent living cells with uneven MSC distribution, i.e. cells remaining mostly in the periphery of the scaffolds (supplementary material 3).

Fig.

Display of a) Acropora and b) Porites scaffolds accessed by microCT: Acropora exhibited larger and more irregular pore size; Porites had a more homogeneous structure with smaller pores. The porosity of Acropora was lower than that of Porites.

In vivo evaluation

Scaffold material bioresorption

X-ray longitudinal analysis revealed that while bioresorption of Porites-TEC could be visualised as early as two months post-implantation with the absence of visible granules and was nearly complete at four months post-implantation, only partial bioresorption of Acropora-TEC was observed at four months (Fig. 2). Residual coral quantities and bioresorption rates at four months differed significantly between Acropora- and Porites-TECs: 114 sd 92 mm³versus 5 sd 5 mm³ (p = 0.008), 81 sd 5% versus 94 sd 6%, respectively (p = 0.04)(Fig. 3b). Due to the fast bioresorption of the Porites-TECs, distribution of the scaffold material bioresorption along the defect could only be assessed for Acropora-TEC and it was evenly distributed among the three defect areas. Remaining coral was surrounded by either bone or fibrous tissue (Fig. 4).

Fig. 2

Radiographs after surgery and at two months, CT reconstructions, and histological slides at four months of the metatarsal bone defects of animals implanted with Acropora-tissue-engineered constructs (TEC) a), Porites-TEC b) and autograft c).

At two months post-operatively, on radiographs, newly-formed bone could not be distinguished from the remaining scaffold material in case of Acropora-TEC (a), but partial to full bioresorption was observed with Porites-TEC (b). At 4 months, full bone regeneration was observed in some animals (a and b top), resembling that observed in autografted animals (c). Recorticalisation was observed in the Acropora-TEC filled defect (a top). In the other animals, new bone formation was limited (a and b bottom). There were still Acropora-TEC present in the defect (a), whereas almost no Porites-TEC remained (b), four months post-operatively. Stains: Stevenel blue and von Gieson picrofuschin. Bone, cells, and coral stained red, blue, and brown, respectively.

Fig.

Graphs showing quantitative analysis of new bone formation and scaffold material bioresorption in defects filled with Acropora-tissue-engineered constructs (Acropora-TEC), Porites-TEC and autograft, four months post-operatively. a) The amount of newly formed bone was not statistically different in defects filled with either Acropora- or Porites-TECs (p = 0.09). High variability and scattering of the pertinent values were observed in the Acropora-TEC group. Two of the defects filled with Acropora-TECs showed the greatest amount of newly formed bone and full bone regeneration, (red triangles), these values were higher than those observed with the Porites-TECs (squares) and autograft cases (circles). b) The bioresorption rates of the scaffold material were lower in Acropora-TEC than in Porites-TEC (p = 0.04). c) New bone formation in Acropora- and Porites-TECs was similar based on the area of the defect, except in the proximal third (p = 0.01). The two Acropora-TEC (blue triangles) and the four Porites-TEC (black squares) which exhibited the highest scaffold material bioresorption (more than 90%; grey line) had the least bone formation.

Fig.

Representative histology of newly formed bone in the tested defects. New bone tissue was present above and inside the remaining coral scaffolds (a and b). Both mature and immature bone tissue was observed, with, respectively, well-orientated, small and dark cells (osteocytes in lacunae) forming a lamellar tissue (c and e) and disorganised, large-nucleated cells forming a non-lamellar tissue (b and f). Abundant osteoid (yellow arrow heads) encircled by bone-lining cells (black arrow heads) was present surrounding the bone tissue, revealing active bone formation (c, e and f). When bone tissue was absent, fibrous tissue was filing the defect (d). The images were obtain from two sheep of the Acropora-TEC group.

Stains: Stevenel Blue and von Gieson picrofuschin. Bone, cells, and coral stained red, blue, and brown, respectively.

New bone tissue was present above and inside the remaining coral scaffolds (a, b). Both mature and immature bone tissue was observed, with, respectively, well orientated, small and dark cells (osteocytes in lacunae) forming a lamellar tissue (c, e), and disorganised, large-nucleated cells forming a non-lamellar tissue (b, f). Abundant osteoid (yellow arrow heads), encircled by bone-lining cells (black arrow heads), was present surrounding the bone tissue, revealing active bone formation (c, e, f). When bone tissue was absent, fibrous tissue filled the defect (d). The images were obtained from two sheep of the Acropora-TEC group. Stains: Stevenel blue and von Gieson picrofuschin. Bone, cells and coral stained red, blue and brown, respectively.

Bone formation

Early bone formation in Acropora-TECs was difficult to evaluate using radiographs because slow scaffold material bioresorption prevented distinction between the newly formed bone and the remaining substrate scaffold (Fig. 2). Similarly, with autograft, bone healing was not assessed. In contrast, bone formation could be observed as early as two months post-implantation on fast-resorbing Porites-TECs (Fig. 2).

At four months post-implantation, the results were highly variable in both TEC groups (Fig. 2). Bone deposition was either: scarce and confined to the bone edges (three of seven animals with Acropora-TECs, three of six with Porites-TECs); nonunion with defects mostly filled with fibrous tissue occurred in six of the tested animals (Fig. 4); or abundant and present at a distance from the bone edges (union of at least one cortical site in four of seven animals with Acropora-TECs and in three of six animals with Porites-TECs). Bone formation permitted full bone regeneration, defined as four cortices united, in two of four united defects with Acropora-TECs and one of three united with Porites-TECs. Moreover, remodeling with recorticalisation was observed in one of the animals with Acropora-TEC. Taken together, these findings suggest that bone union was more frequent with Acropora-TECs.

The occurrence of bone union affected the distribution of the newly formed bone, regardless of the TEC tested. When bone union was achieved, bone was uniformly distributed between the external (in the continuity of the cortices) and the inner (in the continuity of the medullary canal) parts of the defect. In contrast, when nonunion occurred, bone was limited to the inner part, with no bone tissue observed at the external part.

Histological analysis revealed in all examined sections that, irrespective of the TEC tested, mature and immature bone, osteoid, osteocytes, osteoblasts and bone-lining cells were present both in contact with the remaining coral scaffold (even in the core of the coral cubes) and in the bone defect edges (Fig. 4).

When compared with Porites-TECs, Acropora-TECs achieved a two-fold increase in the volume of newly formed bone (1437 sd 1089 mm³versus 782 sd 507 mm³) (Fig. 3a). However, this trend did not show statistical significance (p = 0.09). In addition, two Acropora-TECs exhibited a larger amount of newly formed bone compared with all Porites-TECs and with animals that received autograft (1960 sd 518 mm³).

In all groups, newly formed bone was similarly distributed in the (in the central area of the defect and in the proximal and distal bone defect areas) (Fig. 3c). When comparing the two TECs, distribution of the newly formed bone was similar in the central and distal areas of the defect, but in the case of Acropora-TECs, the rate of newly formed bone was significantly higher than that observed in Porites-TECs in the proximal area (p = 0.01).

Bone formation/scaffold bioresorption coupling

In our study, Acropora-TECs exhibited a slower rate of bioresorption than Porites-TECs and tended to result in more bone formation, suggesting an inverse coupling between the two processes. At four months, the bioresorption rates of the two best performing Acropora-TECs and Porites-TECs were 64% and 86%, and 85% and 87%, respectively (Fig. 3). The two Acropora-TECs and the four Porites-TECs which exhibited the highest rate of scaffold material bioresorption (more than 90%) presented the lowest rate of bone formation. These findings showed that there was no correlation between bone formation and scaffold bioresorption with either TEC, but premature scaffold material bioresorption impaired bone healing in many cases.