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Bone & Joint Research
Vol. 6, Issue 4 | Pages 208 - 215
1 Apr 2017
Decambron A Manassero M Bensidhoum M Lecuelle B Logeart-Avramoglou D Petite H Viateau V

Objectives

To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects.

Materials and Methods

Bone marrow-derived, autologous MSCs were seeded on Acropora or Porites coral granules in a perfusion bioreactor. Acropora-TECs (n = 7), Porites-TECs (n = 6) and bone autografts (n = 2) were then implanted into 25 mm long metatarsal diaphyseal defects in sheep. Bimonthly radiographic follow-up was completed until killing four months post-operatively. Explants were subsequently processed for microCT and histology to assess bone formation and coral bioresorption. Statistical analyses comprised Mann-Whitney, t-test and Kruskal–Wallis tests. Data were expressed as mean and standard deviation.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_1 | Pages 114 - 114
1 Jan 2017
Decambron A Fournet A Manassero M Bensidhoum M Logeart-Avramoglou D Petite H Viateau V
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Bone tissue engineering constructs (BTEC) combining natural resorbable osteoconductive scaffolds and mesenchymal stem cells (MSCs) have given promising results to repair critical size bone defect. Yet, results remain inconsistent. Adjonction of an osteoinductive factor to these BTEC, such as rh-BMP-2, to improve bone healing, seems to be a relevant strategy. However, currently supraphysiological dose of this protein are used and can lead to adverse effects such as inflammation, ectopic bone and/or bone cyst formation. Interestingly, in a preliminary study conducted in ectopic site in a murine model, a synergistic effect on bone formation was observed only when a low dose of rh-BMP-2 was associated with MSCs-seeded coral scaffolds but not with a high dose.

The objective of the study was then to evaluate a BTEC combining coral scaffold, MSCs and a low dose of rh-BMP-2 in a large animal model of clinical relevance.

Sixteen sheep were used for this study.

MSCs were isolated from an aspirate of bone marrow harvested from the iliac crest of each sheep receiving BTEC with MSCs, cultivated and seeded on Acroporacoral scaffolds one week before implantation.

Rh-BMP-2, used at two different doses (low dose: 68μg/defect and high dose: 680μg/defect), was diluted and absorbed on Acroporacoral scaffold one day before implantation.

Metatarsal segmental bone defects (25 mm) were made in the left metatarsal bone of the sheep, stabilized by plate fixation, and filled with Acroporacoral scaffolds loaded with either (i) MSCs and a low dose of rh-BMP-2 (Group 1;n=6), (ii) a low dose of rh-BMP-2 (Group 2;n=5), (iii) a high dose of rh-BMP-2 (Group 3;n=5). Standard radiographs were taken after each surgery and each month until sheep sacrifice, 4 months postoperatively. Bone healing and scaffold resorption were assessed by micro-computed-tomography (μCT) and histomorphometry. Results were compared to a historical control group in which coral scaffolds were loaded with MSCs.

Bone volumes (BV) evaluated by μCT and bone surfaces (BS) evaluated by histomorphometry did not differ between groups (BV: 1914±870, 1737±841, 1894±1028 and 1835±1342 mm3; BS: 25,41±14,25, 19,85±8,31, 25,54±16,98 and 26,08±22,52 %; groups 1, 2, 3 and control respectively); however, an higher bone union was observed in group 1 compared to the others (3, 1, 2 and 2 sheep with bone union in groups 1, 2, 3 and control respectively). No histological abnormalities were observed in any group. Coral resorption was almost complete in all specimens. No significant difference in coral volumes and coral surfaces was observed between groups. A trend towards a higher variability in coral resorption was noted in group 1 compared to the others.

There seems to be a benefit to associate low dose of rh-BMP-2 with MSCs-seeded coral scaffolds as this strategy allowed an increase of bone unions in our model. Yet, results remain inconsistent. Although, defective coupling between scaffold resorption and bone formation impaired bone healing in some animals, adjunction of rh-BMP-2 (even at low dose) to CSMs loaded construct is a promising strategy for bone tissue engineering.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 243 - 243
1 Jul 2014
Decambron A Manassero M Bensidhoum M Petite H Viateau V
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Summary

MSCs could promote bone regeneration in sheep when loaded on natural fully-resorbable scaffolds, but results are highly variable. Improving the ultimate performance of cell-containing constructs cannot be limited to the decreased rate of scaffold resorption.

Introduction. Tissue constructs containing mesenchymal stem cells (MSCs) are an appealing strategy for repairing massive segmental bone defects. However, their therapeutic effectiveness does not match that of autologous bone grafts; among the failure reasons the scaffold resorbability has been identified as a critical feature for achieving bone regeneration. In the present study, the osteogenic potential of 2 constructs obtained by expanding in a bioreactor autologous MSCs onto granules of Acropora or Porites coral, natural fully-resorbable scaffolds, was compared.

Materials and methods

15 sheep underwent a 25 mm long metatarsal ostectomy stabilised with a 3.5 DCP plate. Bone defects were replaced with (i) MSCs-Acropora constructs (n=7), (ii) MSCs-Porites constructs (n=6), (iii) autograft (n=2). Animals were sacrificed 4 months later and bone healing and coral resorption was documented by radiographic, histologic and microCT studies.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 70 - 70
1 Jul 2014
Manassero M Viateau V Decambron A Deschepper M Bensidhoum M Logeart D Petite H
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Summary

Despite similar, early and massive death, hMSCs promote bone formation which was higher in orthotopic than ectopic site suggesting a trophic effect of hMSCs. Ectopic implantation is suitable to evaluate cell survival, but assessment of bone formation requires orthotopic implantation

Introduction

Tissue constructs containing mesenchymal stem cells (MSCs) are appealing strategies for repairing large segmental bone defects but they do not allow consistent bone healing and early and massive MSCs death was identified as a cause of failure. However, little is known about cell survival in the clinical micro-environment encountered during bone healing process, whereas ectopic evaluation is well documented.

In vivo, luciferase-labelled human MSCs survival, within osteoconductive scaffold, was compared in orthotopic and ectopic locations, and bone formation ability of LF-hMSCs-Acropora constructs was evaluated. Interest and limits of each model were highlighted.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 114 - 114
1 Jul 2014
Viateau V Manassero M Petite H Logeart-Avramoglou D Sladkova M Oudina K Bensidhoum M
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Short Summary

The present study demonstrated the feasibility of culturing a large number of standardised granular MSC-containing constructs in a packed bed/column bioreactor that can produce sheep MSC-containing constructs to repair critical-size bone defects in sheep model.

Introduction

Endogenous tissue regeneration mechanisms do not suffice to repair large segmental long-bone defects. Although autologous bone graft remains the gold standard for bone repair, the pertinent surgical technique is limited. Tissue constructs composed of MSCs seeded onto biocompatible scaffolds have been proposed for repairing bone defects and have been established in clinically-relevant animal models. Producing tissue constructs for healing bone defects of clinically-relevant volume requires a large number of cells to heal an approximately 3 cm segmental bone defect. For this reason, a major challenge is to expand cells from a bone marrow aspirate to a much larger, and sufficient, number of MSCs. In this respect, bioreactor systems which provide a reproducible and well-controlled three-dimensional (3D) environment suitable for either production of multiple or large size tissue constructs are attractive approaches to expand MSCs and obtain MSC-containing constructs of clinical grade. In these bioreactor systems, MSCs loaded onto scaffolds are exposed to fluid flow, a condition that provides both enhanced access to oxygen and nutrients as well as fluid-flow-driven mechanical stimulation to cells. The present study was to evaluate bioreactor containing autologous MSCs loaded on coral scaffolds to repair critical-size bone defects in sheep model.


Orthopaedic Proceedings
Vol. 93-B, Issue SUPP_IV | Pages 532 - 532
1 Nov 2011
Lesaichot V Sailhan F Leperlier D Viateau V Petite H
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Purpose of the study: The process of bone lengthening involves three phases: a latence period, distraction, then healing. The healing phase required stability maintained by an external fixator (EF) for 1.16 months/cm lengthening. This time exposes the patient to serious complications. The objective is to accelerate the healing phase in order to shorten the time the patient has to wear the EF. The effect of BMP on osteogenesis in distraction remains a controversial issue. This work was conducted to evaluate the benefit provided by rhBMP-2 for healing the regenerate bone after distraction.

Material and methods: Thirty-nine subadult male rabbits were selected at random. On day 0, a tibial osteotomy was performed followed by installation of a M101 EF. After the latency period of seven days, the distraction began at the rate of 0.5mm/12 h for 21 days. At day 28, at the end of distraction, a new operation was performed and three groups of 13 individuals were created at random. The first group received no material, the second a collagen type 1 sponge, and the third group a collagen type 1 sponge soaked in 100 μg/kg rhBMP-2. The animals were monitored with x-rays, absorptiometry and ultrasound for the qualitative and quantitative analysis. Histological and biomechanical analyses were performed at two months.

Results: Our complication rate was 41%. Qualitative analysis of the x-rays showed, in group 3, the development of more or less voluminous and dense, sometimes hypertrophic calluses. The progression curves of the bone mineral content showed higher values in group 3. The bone mineral content curves remained nevertheless parallel for the three groups. The calluses were thus denser in group 3 but with an early peak density. Groups 1 and 2 had equivalent radiographic and absorptiometric results. The statistical analysis of the imaging findings is ongoing. The histology and biomechanical exams are being performed.

Discussion: The preliminary results show that rhBMP-2 used early in the healing phase enables formation of more dense and hypertrophic calluses. rhBMP-2 does not acceleration the rate of callus formation but stimulates its mineralization. Use of a collagen sponge alone had no effect on healing. Analysis of the histological and mechanic properties observed in the three groups will provide a more precise description of the hypertrophic and strongly mineralized calluses.

Conclusion: Our early results show superior bone mineralization in the treated group.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_II | Pages 254 - 254
1 Jul 2008
VIATEAU V GUILLEMIN G CALANDO Y OUDINA K SEDEL L HANNOUCHE D PETITE H
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Purpose of the study: The objective of this study was to establish an experimental sheep model for a surgical procedure which has been clinically successful for repairing major loss of bone stock: the Masquelet technique.

Material and methods: A 25 mm bone defect was created in a metatarsal bone then filled with a cement filler. After six weeks, the cement was removed after opening the neoformed pseudosynovial membrane. The cavity was left empty in group 1 (n=6) or filled with a morcelized cancellous autograft harvested from the iliac crests in group 1 (n=6).

Results: The surgery was well tolerated in all animals which were able to used the injured limb the day after the operation. Radiographic images and histological findings 24 weeks after surgery demonstrated that healing had not been achieved in all of the animals in group 1. Inversely, healing was achieved in all animals in group 2 at 24 weeks. Immunohistochemistry of the neoformed pseudosynovial demonstrated :

an abundant vascular network,

presence of cells expressing transcription factor CBFA1,

very few inflammatory CD14+ cells (macrophages),

an extracellular matrix positive for type I collagen.

Conclusion: The sheep metatarsal model is a model of critical size with low morbidity. This model could be used to:

evaluate new therapeutic strategies for bone regeneration in conditions close to clinical situations,

study the role of the membrane in bone repair.

The presence of a pseudosynovial membrane might:

be a barrier against the diffusion of bone morphogenetic proteins outside the lesion and

potentially be a reservoir of stem and vascular cells which could be useful for new technologies.