Devakara R. Epari

A Tissue Engineering Solution for Segmental Defect Regeneration in Load-Bearing Long Bones

A polycaprolactone-tricalcium phosphate scaffold with recombinant human BMP-7 heals critical-sized bone defects in sheep. Building Up Bone Large gaps or defects in bone are typically bridged using segments of bone from elsewhere in the body [referred to as autologous bone grafts (ABGs)]. It is not ideal, however, to harvest bone tissue from elsewhere; it is two surgeries, two defect sites, and therefore an increased risk of infection. Instead, tissue engineers have taken on this challenge of replenishing lost bone. In this issue, Reichert and colleagues have designed a polymer-based scaffold that can be loaded with cells and growth factors and inserted directly into a bone defect, with healing demonstrated in sheep after only 3 months. Reichert et al. used their medical-grade polycaprolactone–tricalcium phosphate (mPCL-TCP) scaffolds either alone or in combination with donor mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). The scaffolds were implanted into critical-sized defects (3 cm) in the long bones of sheep, whose bones resemble formation and structure in humans, and are therefore a good model for bone tissue regeneration. After 3 months, the authors reported bone bridging in 100% of the ABGs and scaffold/rhBMP-7 groups but saw bridging in only 38% of the bare scaffold and scaffold/MSC groups. After 12 months, however, animals treated with the scaffold/rhBMP-7 combination showed greater bone volume and mechanical strength than the ABG positive control. The authors attribute this improvement over time to be the result of local BMP delivery (greater stimulation of bone formation) in addition to more bone deposition along the periphery of the defect (enhanced strength). The addition of MSCs did not help bone regeneration, as other studies have shown previously. The next step is determining the ideal BMP dose and the mechanism underlying the effects of the scaffold/rhBMP-7 on surrounding cells and tissue. Then, the hope is to move to clinical trials, where this scaffold will be put to the test for evaluation of bone regeneration and load bearing in humans. The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow–derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep—a model closely resembling human bone formation and structure—were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Bone Regeneration Based on Tissue Engineering Conceptions - A 21st Century Perspective.

The role of Bone Tissue Engineering in the field of Regenerative Medicine has been the topic of substantial research over the past two decades. Technological advances have improved orthopaedic implants and surgical techniques for bone reconstruction. However, improvements in surgical techniques to reconstruct bone have been limited by the paucity of autologous materials available and donor site morbidity. Recent advances in the development of biomaterials have provided attractive alternatives to bone grafting expanding the surgical options for restoring the form and function of injured bone. Specifically, novel bioactive (second generation) biomaterials have been developed that are characterised by controlled action and reaction to the host tissue environment, whilst exhibiting controlled chemical breakdown and resorption with an ultimate replacement by regenerating tissue. Future generations of biomaterials (third generation) are designed to be not only osteoconductive but also osteoinductive, i.e. to stimulate regeneration of host tissues by combining tissue engineering and in situ tissue regeneration methods with a focus on novel applications. These techniques will lead to novel possibilities for tissue regeneration and repair. At present, tissue engineered constructs that may find future use as bone grafts for complex skeletal defects, whether from post-traumatic, degenerative, neoplastic or congenital/developmental “origin” require osseous reconstruction to ensure structural and functional integrity. Engineering functional bone using combinations of cells, scaffolds and bioactive factors is a promising strategy and a particular feature for future development in the area of hybrid materials which are able to exhibit suitable biomimetic and mechanical properties. This review will discuss the state of the art in this field and what we can expect from future generations of bone regeneration concepts.

Timely Fracture-Healing Requires Optimization of Axial Fixation Stability

BACKGROUND Bone-healing is known to be sensitive to the mechanical stability of fixation. However, the influence on healing of the individual components of fixation stiffness remains unclear. The aim of this study was to investigate the relationship between the initial in vitro fixation stiffness and the strength and stiffness of the callus after nine weeks. We hypothesized that axial stiffness would determine the healing outcome. METHODS A standardized midshaft osteotomy of the right tibia was performed on Merino-mix sheep and was stabilized with either one of four monolateral external fixators or one of two tibial nails inserted without reaming. The in vitro stiffness of fixation was determined in six loading conditions (axial compression, torsion, as well as bending and shear in the anteroposterior and mediolateral planes) on ovine tibial specimens. Stiffness was calculated by relating displacements of the fracture fragments, determined by means of attached optical markers, and the loads applied by a materials testing machine. Torsional testing until failure of the explanted tibiae was performed with use of a standard materials testing machine after nine weeks of healing to determine the failure moment and the torsional stiffness of the healed tibia. RESULTS External fixation in sheep generally resulted in higher fixation stiffness than did conventional unreamed tibial nailing. The use of angle-stable locking screws in tibial nailing resulted in fixation stiffness comparable with that of external fixation. The highest torsional moment to failure was observed for the external fixator with moderate axial stiffness and high shear stiffness. The fixator with the highest axial stability did not result in the highest failure moment. Low axial stability in combination with low shear stability resulted in the lowest failure moment. CONCLUSIONS In this study, a clear relationship between the stability of fixation and the mechanical strength of the healing tibia was seen. Moderate levels of axial stability were associated with the highest callus strength and stiffness.

Influence of Scaffold Stiffness on Subchondral Bone and Subsequent Cartilage Regeneration in an Ovine Model of Osteochondral Defect Healing

Background In osteochondral defects, subchondral bone, as a load-bearing structure, is believed to be important for bone and cartilage regeneration. Hypothesis A stiff scaffold creates better conditions for bone formation and cartilage regeneration than does a softer one. Study Design Controlled laboratory study. Methods Critical osteochondral defects were created in the femoral condyles of 24 sheep. Subchondral bone was reconstructed with a stiff scaffold or a modified softer one, with untreated defects serving as controls. The repair response was evaluated with mechanical, histological, and histomorphometrical techniques at 3 and 6 months postoperatively. Results The elastic modulus of regenerated fibrocartilage over the stiff scaffold tended to be higher than in the soft scaffold group (61 % vs 46% of healthy cartilage) at 3 months. No difference was determined at 6 months; all were well below healthy cartilage. Treated defects showed substantial degradation of the soft scaffold with surrounding sclerotic bone at 3 and 6 months. In contrast, degradation of the stiff scaffold was slower and occurred together with continuous osseous replacement. Conclusion Stiff scaffolds were found to improve bone regeneration. In contrast, soft scaffolds provided less support, and consequently subchondral bone became sclerotic. Although regenerated cartilage formed over the stiff scaffolds at 3 months, and these exhibited better mechanical properties than did the soft scaffold group, the mechanical properties in both treated groups were the same at 6 months, not dissimilar to that of tissue formed in the untreated specimens and inferior to native articular cartilage. Clinical Relevance The results imply that subchondral defect filling in clinical settings advances bone regeneration and should have a comparable stiffness to that of healthy subchondral bone rather than being too flexible. Degradation of resorbable materials and consequently the loss of stiffness may compromise the healing of critical defects.

Autologous vs. allogenic mesenchymal progenitor cells for the reconstruction of critical sized segmental tibial bone defects in aged sheep

Mesenchymal progenitor cells (MPCs) represent an attractive cell population for bone tissue engineering. Their special immunological characteristics suggest that MPCs may be used in allogenic applications. The objective of this study was to compare the regenerative potential of autologous vs. allogenic MPCs in an ovine critical size segmental defect model. Ovine MPCs were isolated from bone marrow aspirates, expanded and cultured with osteogenic medium for 2weeks before implantation. Autologous and allogenic transplantation was performed using the cell-seeded scaffolds and unloaded scaffolds, while the application of autologous bone grafts served as a control group (n=6). Bone healing was assessed 12weeks after surgery by radiology, microcomputed tomography, biomechanical testing and histology. Radiology, biomechanical testing and histology revealed no significant differences in bone formation between the autologous and allogenic groups. Both cell groups showed more bone formation than the scaffold alone, whereas the biomechanical data showed no significant differences between the cell groups and the unloaded scaffolds. The results of the study suggest that scaffold-based bone tissue engineering using allogenic cells offers the potential for an off-the-shelf product. Thus the results of this study serve as an important baseline for translation of the assessed concepts into clinical applications.

Pressure, oxygen tension and temperature in the periosteal callus during bone healing-An in vivo study in sheep

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

Size and habit of mineral particles in bone and mineralized callus during bone healing in sheep.

Bone healing is known to occur through the successive formation and resorption of various tissues with different structural and mechanical properties. To get a better insight into this sequence of events, we used environmental scanning electron microscopy (ESEM) together with scanning small‐angle X‐ray scattering (sSAXS) to reveal the size and orientation of bone mineral particles within the regenerating callus tissues at different healing stages (2, 3, 6, and 9 weeks). Sections of 200 µm were cut from embedded blocks of midshaft tibial samples in a sheep osteotomy model with an external fixator. Regions of interest on the medial side of the proximal fragment were chosen to be the periosteal callus, middle callus, intercortical callus, and cortex. Mean thickness (T parameter), degree of alignment (ρ parameter), and predominant orientation (ψ parameter) of mineral particles were deduced from resulting sSAXS patterns with a spatial resolution of 200 µm. 2D maps of T and ρ overlapping with ESEM images revealed that the callus formation occurred in two waves of bone formation, whereby a highly disordered mineralized tissue was deposited first, followed by a bony tissue with more lamellar appearance in the ESEM and where the mineral particles were more aligned, as revealed by sSAXS. As a consequence, degree of alignment and mineral particle size within the callus increased with healing time, whereas at any given moment there were structural gradients, for example, from periosteal toward the middle callus.

Mechanobiology of bone healing and regeneration: in vivo models

Abstract Mechanical boundary conditions are well known to influence the regeneration of bone and mechanobiology is the study of how mechanical or physical stimuli regulate biological processes. In vivo models have been applied over many years to investigate the effects of mechanics on bone healing. Early models have focused on the influence of mechanical stability on healing outcome, with an interest in parameters such as the magnitude of interfragmentary movement, the rate and timing of application of micromotion and the number of loading cycles. As measurement techniques have been refined, there has been a shift in orders of magnitude from investigations targeted at the organ level to those targeted at the tissue level and beyond. An understanding of how mechanics influences tissue differentiation during repair and regeneration crucially requires spatial and temporal knowledge of both the local mechanical environment in the healing tissue and a characterization of the tissues formed over the course of regeneration. Owing to limitations in the techniques available to measure the local mechanical conditions during repair directly, simulation approaches, such as the finite element method, are an integral part of the mechanobiologists toolkit, while histology remains the gold standard in the characterization of the tissue formed. However, with rapid advances occurring in imaging modalities and methods to characterize tissue properties, new opportunities exist to better understand the role of mechanics in the biology of bone regeneration. Combined with developments in molecular biology, mechanobiology has the potential to offer exciting, new regenerative treatments for bone healing.

Biaxial cell stimulation: A mechanical validation

To analyse mechanotransduction resulting from tensile loading under defined conditions, various devices for in vitro cell stimulation have been developed. This work aimed to determine the strain distribution on the membrane of a commercially available device and its consistency with rising cycle numbers, as well as the amount of strain transferred to adherent cells. The strains and their behaviour within the stimulation device were determined using digital image correlation (DIC). The strain transferred to cells was measured on eGFP-transfected bone marrow-derived cells imaged with a fluorescence microscope. The analysis was performed by determining the coordinates of prominent positions on the cells, calculating vectors between the coordinates and their length changes with increasing applied tensile strain. The stimulation device was found to apply homogeneous (mean of standard deviations approx. 2% of mean strain) and reproducible strains in the central well area. However, on average, only half of the applied strain was transferred to the bone marrow-derived cells. Furthermore, the strain measured within the device increased significantly with an increasing number of cycles while the membranes Youngs modulus decreased, indicating permanent changes in the material during extended use. Thus, strain magnitudes do not match the system readout and results require careful interpretation, especially at high cycle numbers.

Stress Shielding in Box and Cylinder Cervical Interbody Fusion Cage Designs

Study Design. A numerical analysis of stress shielding of the bone graft in box and cylinder interbody fusion cages was performed. Objectives. To evaluate the stress shielding characteristics of box and cylinder interbody fusion cages for the cervical spine with regard to their rigidity and contiguous pore size. Summary of Background Data. Cage design has been shown to influence loading of the augmented bone graft tissue. In addition, a large contiguous pore design is believed to be important to avoid stress shielding effects. Methods. A two-dimensional axisymmetric, biphasic finite-element model of the cage incorporating the bone graft and the adjacent vertebral bodies was developed. Analysis was performed in two parts. First, the vertebrae were loaded by an axial compressive force, and second, the effect of vertebral penetration by the interbody cage was simulated. Results. Straining of bone graft in the box cage was generally lower than that of the cylinder cage. The strains in the cylinder cage were seen to be more uniformly distributed, whereas in the box cage straining was concentrated in the graft under the endplates. Vertebral penetration by the cylinder cage resulted in significant straining of the bone graft (28% strain), whereas lower strains were determined in the box cage (a maximum of 17% strain). Conclusions. The central pore in the box design does not seem as effective as the fully open cylinder cage in transferring loads to the augmented graft tissue. Early penetration of the adjacent vertebrae by the cylinder cage may provide early postoperative stability and load the graft tissue, thereby imparting the necessary signals for fusion.