Various mathematical models represent the effects of local mechanical environment on the regulation of skeletal regeneration. Their relevance relies on an accurate description of the evolving mechanical properties of the regenerating tissue. The object of this study was to develop an experimental model which made it possible to characterize the temporal evolution of the structural and mechanical properties during unloaded enchondral osteogenesis in the New Zealand rabbit, a standard animal model for studies of osteogenesis and chondrogenesis. A 25mm segment of tibial diaphysis was removed sub-periosteally from rabbits. The defect was repaired by the preserved periosteum. An external fixator was applied to prevent mechanical loading during osteogenesis. The regenerated skeletal tissues were studied by CT scan, histology and mechanical tests. The traction tests between 7 to 21 days post-surgery were done on formaldehyde-fixated tissue allowing to obtain force/displacement curves. The viscoelastic properties of the regenerating skeletal tissues were visualized throughout the repair process.
Deep Dive into Temporal evolution of mechanical properties of skeletal tissue regeneration in rabbits. An experimental study.
Various mathematical models represent the effects of local mechanical environment on the regulation of skeletal regeneration. Their relevance relies on an accurate description of the evolving mechanical properties of the regenerating tissue. The object of this study was to develop an experimental model which made it possible to characterize the temporal evolution of the structural and mechanical properties during unloaded enchondral osteogenesis in the New Zealand rabbit, a standard animal model for studies of osteogenesis and chondrogenesis. A 25mm segment of tibial diaphysis was removed sub-periosteally from rabbits. The defect was repaired by the preserved periosteum. An external fixator was applied to prevent mechanical loading during osteogenesis. The regenerated skeletal tissues were studied by CT scan, histology and mechanical tests. The traction tests between 7 to 21 days post-surgery were done on formaldehyde-fixated tissue allowing to obtain force/displacement curves. The vis
In the literature, skeletal tissue regeneration has been extensively studied. Its general process involves mesenchymal precursor cells ( 1 ). which first proliferate to fill the tissue volume ( 2 ), then differentiate into mature tissue endowed with remodelling properties ( 3;4 ). These cascades of cellular and molecular events follow programmed time sequences modulated by environmental factors. Among them, biological signals and the local mechanical environment interact with each step of the cascade of cell events involved in tissue regeneration. These interactions led to the theoretical concepts of mechanobiology ( 4;5 ). The stress and strains applied to the precursor cells induce specific biological responses at all levels of the regenerating skeletal structure, from the molecular level of its extra-cellular matrix ( 6;7 ) to its macroscopic morphology ( 4;8 ).
Various numerical model studies have been used to analyse the temporal evolution of mechanical properties of multipotent mesenchymal tissue ( 4; [9][10][11]. Two types of mechanical behaviour have been considered: elastic ( 4;5;12;13 ); and poroelastic ( 4;5;9-13 ). These models have been confirmed by experiments which are not realised on a regenerating tissue.
The New Zealand white rabbit is a standard experimental model for study of osteogenesis and chondrogenesis. However, the temporal evolution of the mechanical properties of these regenerating tissues has not been studied. This work aims to characterize the tensile properties of regenerating skeletal tissue at 1, 2 and 3 weeks in a rabbit tibial defect model.
Our experimental model of skeletal tissue regeneration is based on periosteal properties.
Periosteal stripping from the bone surface induces a cascade of cellular and molecular events which brings mesenchymal precursor cells to the surgical bed ( 14). They will then proliferate before differentiating.
The in vitro test measured the temporal evolution of the mechanical behaviour of this skeletal tissue from the mesenchymal stages until ossification into primary bone. CT scan and histological study completed the traction tests.
Fourteen three-month old New Zealand rabbits weighing 2.5 kg and skeletally immature were used as animal models (INRA-ENSA Montpellier France). The surgery was performed in an accredited experimental surgery laboratory of Montpellier Medical School, in accordance with French regulations on animal care and use of laboratory animals. Two rabbits died during anesthesia; however none of the animals suffered postoperative complication such as pin tract or wound infection. In the postoperative period, the animals ate and walked normally. No animal died postoperatively.
The anesthesia was obtained by premedication with acepromazine 1%, (Labo. CEVA) 10 mg IM, followed by IV continuous infusion of 2% xylazine (Labo. BAYER) 0.27 mg/mn, and 5% ketamine (Labo. MERIAL ) 1.14 mg/mn. Under strict surgical aseptic conditions the medial third of one tibia was exposed via a medial approach. The periosteal sheet was incised longitudinally on the lateral side of the tibia in order to preserve its vascular connections with the saphenous bundle. The periosteum was elevated from the entire circumference of the bone segment. In order to maintain the leg length and axis, a mono-lateral external fixator (Orthofix M 111) was applied and secured to the bone by 4 half pins (diameter 2.5 mm). A 25 mm long bone segment was cut off and removed from the medial third of the tibia. The periosteal mantel was carefully closed back over the segmental bone loss covering the edge of the bone section in order to initiate the skeletal regeneration and act as a barrier against adjacent soft tissue interposition.
The skin was closed around the pins on the medial side off the hind limb. After surgery the animal was restricted to ambulation in its cage. The external fixator allowed the bypass of the mechanical load through the regenerating tissues during the locomotion of the animals (Figure 1).
After the prescribed healing time, the rabbits were sacrificed by an overdose of pentobarbital at chronological dates: Day 7, 14 and 21. The operated tibiae were explanted in order to analyse the regenerated tissues at these three stages of maturation. The tissue samples included the regenerated zones (25 mm) in continuity with their bone attachments on either side, from the knee joint up to 1 cm beyond the most distal pin fixation (Figure 2). Twelve regenerating tibiae (5 obtained at 7 days, 4 at 14 days, 3 at 21 days) and one healthy bone were available for mechanical analysis.
Longitudinal CT scan images of the regenerating tissue samples were obtained (Light speed, General Electric, 0.6 mm between each image). Imaging showed the shape and contours of the regenerating structures and illustrated the evolution of their mineralization. The time required for image acquisition did not exceed five minutes, thus preventing dehydration of the tissues.
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