A Natural Granular Resorbable Scaffold for Bone Reg ... · A Natural Granular Resorbable Scaffold...
1
A Natural Granular Resorbable Scaffold for Bone Regeneration in Sheep + 1,2 Manassero, M; 1,2 Viateau, V; 1,2 Vallefuoco, R; 2 Oudina, K; 2 Valentin, M; 2 Petite, H; 2 Bensidhoum, M + 1 Ecole Nationale Vétérinaire d’Alfort, Maisons Alfort, France; + 2 Laboratoire de Biomécanique et Biomatériaux Ostéoarticulaires UMR 7052, Paris, France [email protected] INTRODUCTION: The possibility of isolating and expanding autologous mesenchymal stem cells (MSC) from a small sample of bone marrow and driving them towards an osteogenic phenotype has led to development of bioengineered bone constructs. In a previous study of a clinically relevant animal model it has been proved that standardized biohybrids engineered from Porites granular coralline scaffolds can be used to efficiently repair large bone defects. However, the quantity of bone formed in defects filled with biohybrids, varied among animals and bone union did not consistently occur. Several authors have emphasized that balanced scaffold resorption and new bone apposition must be obtained for bone healing with both natural and synthetic ceramic scaffolds. Most importantly, it is not yet understood whether the resorption of the scaffold itself is mandatory to trigger bone formation by cells. To address this question, bioengineered bone constructs made of Acropora coral which have less resorbability properties than Porites coral were implanted with or without MSC in a sheep metatarsal segmental critical size defect. MATERIALS AND METHODS: Animals : Nine two-year-old, female sheep were used for this study. All experimental procedures were performed according to the guidelines published by the European Committee for Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Lariboisière Medical Faculty. Scaffold : Acropora coral scaffolds (mean pore diameter of 500 μm; porosity of 12%) were available as cubes of 3x3x3 mm 3 . MSC Isolation and expansion : Bone marrow was aspirated from the iliac crest of each sheep. Non-adherents cells were discarded during media change, after 3 days of culture. Medium was changed three times a week. When the cells were confluent, MSC were detached and were plated (passage 1) at the density of 3x10 3 cells/cm 2 . At 80-90% confluence MSC were detached and cryopreserved. Cell loading of scaffolds : One week before implantation, cryopreserved cells were thawed and washed, 10 5 MSC were seeded per cube of Acropora. And the cell constructs were placed in an incubator for 7 days. Surgical procedure : Osteoperiosteal segmental (25 mm) defects were made in the left metatarsal bone of 9 sheep, stabilized by plate fixation, and replaced with a PMMA spacer. The PMMA spacer was removed 6 weeks later, and the defect was filled either with Acropora coral scaffolds (Group1; n=4); or with Acropora coral scaffolds loaded with MSC (Group2; n=5). Postoperative Management : Postoperative analgesia was provided by intravenous injections of meloxicam and tramadol administered 30 min before the end of each surgical procedure and again 72 h after the procedure. The lower limb was fitted with a short cylindrical cast. In-vivo and post-mortem evaluation : Standard radiographs were taken after each surgery and at the end of months 1, 2, 4 and 6. Immediately after euthanasia, casts were removed, and the metatarsal bone excised. Bone explants were collected for processing. Bone healing and scaffold resorption were assessed by micro-computed-tomography (SkyScan™ 1171 microCT) and undecalcified histology. The data were reported as mean ±SD and analyzed by Mann-Whitney test. Values p<0.05 were considered significant. RESULTS: All animals recovered well and stable bone fixation for all animals was obtained throughout the study. Muscle wastage was observed on the operated limb in all sheep as early as 3 weeks postoperatively, suggesting an abnormal weight bearing pattern but this did not differ between Group1 and Group2. By 1 month, radiographs showed resorption of the coral scaffold in all animals but this process was not complete and not the same in all animal. Six months after implantation, animals were sacrificed and each defect was assessed by micro-computed tomography and histology. No bone union was observed in Group1, in contrast to Group2 where bone continuity was observed. Acropora resorption rate was higher in Group1 than in Group2 (99.2±0.8% v.s. 93.0±4.7%) (Figure 1A). The amounts of newly formed bone in defects filled with Acropora/MSC (Group2) were significantly higher from those filled with Acropora alone (Group1) (677±227 v.s. 1357±471 mm 3 ) (Figure 1B). A B Figure 1: Resorption rate (B) and volume of newly bone formed (A) Whereas cell-free implant rarely displayed bone formation (mostly observed in the bony edges vicinity) with an abundant fibrous tissue, a consistent bone formation was observed with MSC, even inner the defect at distance to bony edges (Figure 2). Although the results remained variable among animals, a fully bone regeneration was obtained in one animal of the Group2 (Figure 2). A C B D Figure 2: Representative 3D tomodensitometric reconstruction of sheep belonging to Group1 (A) and Group2 (B), and histology of sections inner the defect of sheep belonging to Group1 (C) and Group2 (D). The histological stain was Stevenel Blue and von Gieson picrofuschin. (Original magnification: x4, red: bone, brown: coral, *: fibrous tissue) DISCUSSION AND CONCLUSION: In the present study, the osteogenic potential of MSC/Acropora coral constructs for repairing long segmental critical-sized bone defects in sheep was investigated. MSC were loaded onto Acropora coral scaffolds which are less resorbable coral than Porites coral used in the previous study. Most importantly, the findings showed that granules of MSC/coral constructs enhanced significantly the rate of new bone formation. High amounts of newly formed bone were detected in defects filled with coral/MSC allowing to obtain a fully bone regeneration in one animal. Interestingly, MSC seems to slightly decrease the resorption rate but this was not significant. Despite a high variability, our results may have important implications for designing bone regeneration strategies based on stem cells; we observed that, in a clinically relevant model, the osteogenic ability of bone constructs based on MSC and a natural resorbable could be used to repair large segmental defects. SIGNIFICANCE: This study established clearly the benefits of using Acropora scaffold loaded with MSC for long bone defect in sheep since this tissue construct allowed to obtained a fully bone regeneration. Further studies are needed to understand inter-individual variability. REFERENCES: Viateau V, et al. Journal of orthopaedic research. 2007;25(6):741-9. Bensidhoum M, et al. 7th Combined Meeting of the Orthopaedic Research Societies; 2010, N°39, Kyoto. * Poster No. 0631 • ORS 2012 Annual Meeting
Transcript of A Natural Granular Resorbable Scaffold for Bone Reg ... · A Natural Granular Resorbable Scaffold...
A Natural Granular Resorbable Scaffold for Bone Regeneration in Sheep
+ 1,2Manassero, M; 1,2Viateau, V; 1,2Vallefuoco, R; 2Oudina, K; 2Valentin, M; 2Petite, H; 2Bensidhoum, M
+1 Ecole Nationale Vétérinaire d’Alfort, Maisons Alfort, France; +2 Laboratoire de Biomécanique et Biomatériaux Ostéoarticulaires UMR 7052, Paris, France [email protected]
INTRODUCTION: The possibility of isolating and expanding autologous mesenchymal stem cells (MSC) from a small sample of bone marrow and driving them towards an osteogenic phenotype has led to development of bioengineered bone constructs. In a previous study of a clinically relevant animal model it has been proved that standardized biohybrids engineered from Porites granular coralline scaffolds can be used to efficiently repair large bone defects. However, the quantity of bone formed in defects filled with biohybrids, varied among animals and bone union did not consistently occur. Several authors have emphasized that balanced scaffold resorption and new bone apposition must be obtained for bone healing with both natural and synthetic ceramic scaffolds. Most importantly, it is not yet understood whether the resorption of the scaffold itself is mandatory to trigger bone formation by cells. To address this question, bioengineered bone constructs made of Acropora coral which have less resorbability properties than Porites coral were implanted with or without MSC in a sheep metatarsal segmental critical size defect. MATERIALS AND METHODS: Animals: Nine two-year-old, female sheep were used for this study. All experimental procedures were performed according to the guidelines published by the European Committee for Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Lariboisière Medical Faculty. Scaffold: Acropora coral scaffolds (mean pore diameter of 500 µm; porosity of 12%) were available as cubes of 3x3x3 mm3. MSC Isolation and expansion: Bone marrow was aspirated from the iliac crest of each sheep. Non-adherents cells were discarded during media change, after 3 days of culture. Medium was changed three times a week. When the cells were confluent, MSC were detached and were plated (passage 1) at the density of 3x103 cells/cm2. At 80-90% confluence MSC were detached and cryopreserved. Cell loading of scaffolds: One week before implantation, cryopreserved cells were thawed and washed, 105 MSC were seeded per cube of Acropora. And the cell constructs were placed in an incubator for 7 days. Surgical procedure: Osteoperiosteal segmental (25 mm) defects were made in the left metatarsal bone of 9 sheep, stabilized by plate fixation, and replaced with a PMMA spacer. The PMMA spacer was removed 6 weeks later, and the defect was filled either with Acropora coral scaffolds (Group1; n=4); or with Acropora coral scaffolds loaded with MSC (Group2; n=5). Postoperative Management: Postoperative analgesia was provided by intravenous injections of meloxicam and tramadol administered 30 min before the end of each surgical procedure and again 72 h after the procedure. The lower limb was fitted with a short cylindrical cast. In-vivo and post-mortem evaluation: Standard radiographs were taken after each surgery and at the end of months 1, 2, 4 and 6. Immediately after euthanasia, casts were removed, and the metatarsal bone excised. Bone explants were collected for processing. Bone healing and scaffold resorption were assessed by micro-computed-tomography (SkyScan™ 1171 microCT) and undecalcified histology. The data were reported as mean ±SD and analyzed by Mann-Whitney test. Values p<0.05 were considered significant. RESULTS: All animals recovered well and stable bone fixation for all animals was obtained throughout the study. Muscle wastage was observed on the operated limb in all sheep as early as 3 weeks postoperatively, suggesting an abnormal weight bearing pattern but this did not differ between Group1 and Group2. By 1 month, radiographs showed resorption of the coral scaffold in all animals but this process was not complete and not the same in all animal. Six months after implantation, animals were sacrificed and each defect was assessed by micro-computed tomography and histology. No bone union was observed in Group1, in contrast to Group2 where bone continuity was observed.
Acropora resorption rate was higher in Group1 than in Group2 (99.2±0.8% v.s. 93.0±4.7%) (Figure 1A). The amounts of newly formed bone in defects filled with Acropora/MSC (Group2) were significantly higher from those filled with Acropora alone (Group1) (677±227 v.s. 1357±471 mm3) (Figure 1B).
A
B
Figure 1: Resorption rate (B) and volume of newly bone formed (A) Whereas cell-free implant rarely displayed bone formation (mostly observed in the bony edges vicinity) with an abundant fibrous tissue, a consistent bone formation was observed with MSC, even inner the defect at distance to bony edges (Figure 2). Although the results remained variable among animals, a fully bone regeneration was obtained in one animal of the Group2 (Figure 2).
A C
B D Figure 2: Representative 3D tomodensitometric reconstruction of sheep
belonging to Group1 (A) and Group2 (B), and histology of sections inner the defect of sheep belonging to Group1 (C) and Group2 (D). The histological stain was Stevenel Blue and von Gieson picrofuschin.
(Original magnification: x4, red: bone, brown: coral, *: fibrous tissue) DISCUSSION AND CONCLUSION: In the present study, the osteogenic potential of MSC/Acropora coral constructs for repairing long segmental critical-sized bone defects in sheep was investigated. MSC were loaded onto Acropora coral scaffolds which are less resorbable coral than Porites coral used in the previous study. Most importantly, the findings showed that granules of MSC/coral constructs enhanced significantly the rate of new bone formation. High amounts of newly formed bone were detected in defects filled with coral/MSC allowing to obtain a fully bone regeneration in one animal. Interestingly, MSC seems to slightly decrease the resorption rate but this was not significant. Despite a high variability, our results may have important implications for designing bone regeneration strategies based on stem cells; we observed that, in a clinically relevant model, the osteogenic ability of bone constructs based on MSC and a natural resorbable could be used to repair large segmental defects. SIGNIFICANCE: This study established clearly the benefits of using Acropora scaffold loaded with MSC for long bone defect in sheep since this tissue construct allowed to obtained a fully bone regeneration. Further studies are needed to understand inter-individual variability. REFERENCES: Viateau V, et al. Journal of orthopaedic research. 2007;25(6):741-9. Bensidhoum M, et al. 7th Combined Meeting of the Orthopaedic Research Societies; 2010, N°39, Kyoto.
*
Poster No. 0631 • ORS 2012 Annual Meeting
![Postoperative Infection Caused by a Resorbable …replace titanium fixation systems in craniofacial, oral, maxillofacial, plastic, and reconstructive surgery [1,2]. The resorbable](https://static.fdocuments.in/doc/165x107/5e8a899e4b672e3ccb0c3507/postoperative-infection-caused-by-a-resorbable-replace-titanium-fixation-systems.jpg)
