Atomic force microscopy of collagen structure in bone and dentine revealed by osteoclastic resorption

Mineralised tissues such as bone consist of two material phases: collagen protein fibrils, secreted by osteoblasts, form model structures for subsequent deposition of mineral, calcium hydroxyapatite. Collagen and mineral are removed in a three-dimensional manner by osteoclasts during bone turnover in skeletal growth or repair. Bone active drugs have recently been developed for skeletal diseases, and there is revived interest in changes in the structure of mineralised tissues seen in disease and upon treatment. The resolution of atomic force microscopy and use of unmodified samples has enabled us to image bone and dentine collagen exposed by the natural process of cellular dissolution of mineralised matrix. The morphology of bone and dentine has been analysed when fully mineralised and after osteoclast-mediated bone resorption, and compared with results from other microscopy techniques. Banded type I collagen, with 66.5+/-1.4 nm axial D-periodicity and 62.2+/-7.0 nm diameter, has been identified within resorption lacunae in bone and 69.4+/-4.3 nm axial D-periodicity and 140.6+/-12.4 nm diameter in dentine substrates formed by human and rabbit osteoclasts, respectively. This observation suggests a route by which the material and morphological properties of bone collagen can be analysed in situ, compared with collagen from non-skeletal sites, and contrasted in diseases of medical importance, such as osteoporosis, where skeletal tissue is mechanically weakened.     

Osteocyte pericellular and perilacunar matrices as markers of bone–implant mechanical integrity

To develop durable bone healing strategies through improved control of bone repair, it is of critical importance to understand the mechanisms of bone mechanical integrity when in contact with biomaterials and implants. Bone mechanical integrity is defined here as the adaptation of structural properties of remodeled bone in regard to an applied mechanical loading. Accordingly, the authors present why future investigations in bone repair and regeneration should emphasize on the matrix surrounding the osteocytes. Osteocytes are mechanosensitive cells considered as the orchestrators of bone remodeling, which is the biological process involved in bone homeostasis. These bone cells are trapped in an interconnected porous network, the lacunocanalicular network, which is embedded in a bone mineralized extracellular matrix. As a consequence of an applied mechanical loading, the bone deformation results in the deformation of this lacunocanalicular network inducing a shift in interstitial fluid pressure and velocity, thus resulting in osteocyte stimulation. The material environment surrounding each osteocyte, the so called perilacunar and pericellular matrices properties, define its mechanosensitivity. While this mechanical stimulation pathway is well known, the laws used to predict bone remodeling are based on strains developing at a tissue scale, suggesting that these strains are related to the shift in fluid pressure and velocity at the lacunocanalicular scale. While this relationship has been validated through observation in healthy bone, the fluid behavior at the bone-implant interface is more complex. The presence of the implant modifies fluid behavior, so that for the same strain at a tissue scale, the shift in fluid pressure and velocity will be different than in a healthy bone tissue. In that context, new markers for bone mechanical integrity, considering fluid behavior, have to be defined. The viewpoint exposed by the authors indicates that the properties of the pericellular and the perilacunar matrices have to be systematically investigated and used as structural markers of fluid behavior in the course of bone biomaterial development.     

Effect of non-enzymatic glycation on collagen nanoscale mechanisms in diabetic and age-related bone fragility

Age and diabetes have long been known to induce an oxidative reaction between glucose and collagen, leading to the accumulation of advanced glycation end-products (AGEs) cross-links in collagenous tissues. More recently, AGEs content has been related to loss of bone quality, independent of bone mass, and increased fracture risk with aging and diabetes. Loss of bone quality is mostly attributed to changes in material properties, structural organization, or cellular remodeling. Though all these factors play a role in bone fragility disease, some common recurring patterns can be found between diabetic and age-related bone fragility. The main pattern we will discuss in this viewpoint is the increase of fibrillar collagen stiffness and loss of collagen-induced plasticity with AGE accumulation. This study focused on recent related experimental studies and discusses the correlation between fluorescent AGEs content at the molecular and fibrillar scales, collagen deformation mechanisms at the nanoscale, and resistance to bone fracture at the macroscale.     

Multiscale characterization of pathological bone tissue

Bone is a complex natural material with a complex hierarchical multiscale organization, crucial to perform its functions. Ultrastructural analysis of bone is crucial for our understanding of cell to cell communication, the healthy or pathological composition of bone tissue, and its three-dimensional (3D) organization. A variety of techniques has been used to analyze bone tissue. This article describes a combined approach of optical, scanning electron, and transmission electron microscopy for the ultrastructural analysis of bone from the nanoscale to the macroscale, as illustrated by two pathological bone tissues. By following a top-down approach to investigate the multiscale organization of pathological bones, quantitative estimates were made in terms of calcium content, nearest neighbor distances of osteocytes, canaliculi diameter, ordering, and D-spacing of the collagen fibrils, and the orientation of intrafibrillar minerals which enable us to observe the fine structural details. We identify and discuss a series of two-dimensional (2D) and 3D imaging techniques that can be used to characterize bone tissue. By doing so we demonstrate that, while 2D imaging techniques provide comparable information from pathological bone tissues, significantly different structural details are observed upon analyzing the pathological bone tissues in 3D. Finally, particular attention is paid to sample preparation for and quantitative processing of data from electron microscopic analysis.     

Influence of anti-inflammatory administration in collagen maturation process during orthodontic tooth movement

Bone formation is essential to orthodontic tooth movement and bone is formed by collagen. To analyze the collagen maturation process on bone matrix neoformed under nonsteroidal and steroidal treatment during orthodontic tooth movement by polarized microscopy, male Wistar rats (n = 90) were randomly divided into three groups (n = 30): C (control), NSAID (potassium diclofenac) and SAID (disodic phosphate dexamethasone). The animals of the C group received 0.9% saline solution; NSAID group received 5 mg/kg potassium diclofenac (CATAFLAM®); and SAID group received 2 mg/kg phosphate dissodic dexamethasone (DEXANIL®). Animals were sacrificed 3, 7 or 14 days after the placement of orthodontic appliances and the upper first molars were processed histologically and stained with picrosirius. Bone formation was evaluated under polarized light microscopy and 4.5 Image Pro‐Plus® software calculated the percentage of immature/mature collagen present in the groups. On the third days after force application, SAID and NSAID groups showed greater proportion of immature collagen than C group. On the seventh and fourteenth days, there was a lower proportion of mature collagen only in the SAID group (P < 0.001). These data demonstrate that dexamethasone delays the collagen maturation process in established bone matrix. Microsc. Res. Tech., 2011. © 2010 Wiley‐Liss, Inc.     

Irregular Shaped,Assumably Semi‐Crystalline Calciumphosphate Platelet Deposition at the Mineralization Front of Rabbit Femur Osteotomy: A HR‐TEM Study

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high‐resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow‐up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex‐shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169‐182, 2013. © 2012 Wiley Periodicals, Inc.     

Helium ion microscopy for high-resolution visualization of the articular cartilage collagen network

The articular cartilage collagen network is an important research focus because network disruption results in cartilage degeneration and patient disability. The recently introduced helium ion microscope (HIM), with its smaller probe size, longer depth of field and charge neutralization, has the potential to overcome the inherent limitations of electron microscopy for visualization of collagen network features, particularly at the nanoscale. In this study, we evaluated the capabilities of the helium ion microscope for high-resolution visualization of the articular cartilage collagen network. Images of rabbit knee cartilage were acquired with a helium ion microscope; comparison images were acquired with a field emission scanning electron microscope (FE-SEM) and a transmission electron microscope (TEM). Sharpness of example high-resolution helium ion microscope and field emission scanning electron microscope images was quantified using the 25-75% rise distance metric. The helium ion microscope was able to acquire high-resolution images with unprecedented clarity, with greater sharpness and three-dimensional-like detail of nanoscale fibril morphologies and fibril connections, in samples without conductive coatings. These nanoscale features could not be resolved by field emission scanning electron microscopy, and three-dimensional network structure could not be visualized with transmission electron microscopy. The nanoscale three-dimensional-like visualization capabilities of the helium ion microscope will enable new avenues of investigation in cartilage collagen network research.     

Image analysis of mineralized and non-mineralized type I collagen fibrils

Turkey leg tendons at an early stage of mineralization have been thin sectioned and imaged by electron microscopy. At this stage collagen-associated mineral apatite was found to be present within both the gap and overlap zones. The earliest apatite occurs in a microcrystalline form which gives a rather generalized and characteristic density to both the gap and overlap zones; with subsequent development larger defined apatite crystals arise which span gap/overlap zones. Fourier transformation of such images revealed the major 67 nm axial repeat of the gap/overlap zone plus four other maxima corresponding to repeat spacings of 22, 16, 13, and 11 nm respectively. Computer imaging techniques were used to reconstruct images by using selected spatial frequencies from such transforms. In this manner the subperiodic distributions of mineral were visually enhanced. These subperiodicities are positioned in an asymmetric fashion over the entire D unit repeat aligning with the molecular orientation of the fibril. Analyses of both negatively stained collagen and computer-generated maps of collagen hydrophobicity were compared to the mineral distribution of collagen. Densitometric comparisons showed a positional correlation between the axial banding patterns of mineralized fibrils and those of negatively stained non-mineralized fibrils. Comparable spatial frequencies were also present in transforms between hydrophobic maps and mineral distribution of collagen. These results suggest that the lateral clusterings of hydrophobic residues which span the fibril at specific sites in both the gap and overlap zones serve to prohibit early mineral deposition. This observed hydrophobic influence in combination with the gap space appear as contributing factors in the observed axial distribution of mineral within collagen.     

Compressive mechanical properties of bovine cortical bone under varied loading rates

Bone tissue functions in varied mechanical systems of the body under static and dynamic conditions. Therefore, it is essential to understand the mechanical responses of bone at varied loading rates, especially those at fast loading rates. This study has investigated the effect of loading rate on the compressive mechanical properties of bovine cortical bone. Bone specimens of 3.85 mm in diameter and 7.7 mm in length were compressed longitudinally with the loading rates of 2 to 2000 mm/s (corresponding strain rates of 0.26 to 260 s(-1)). As a result, bovine cortical bone showed high linear elasticity when the loading rate was slow, and exhibited three definite regions of linear elasticity, plastic deformation, and densification at faster loading rates. The elastic modulus showed no dependency on the loading rate. Compressive strength, strain at fracture, and toughness increased as the loading rate increased under the condition that the loading rates were slower than each critical loading rate of 1000, 100, and 1500 mm/s, respectively. However, all showed no significant changes when the loading rates were faster than the corresponding critical loading rates. In conclusion, as the loading rate increased, changes in the compressive mechanical parameters were different depending on the parameter and the loading rate range. Compressive mechanical behaviour of bovine cortical bone showed a brittle nature under high strain rates (strain rates > 13 s(-1)). These findings should be reflected in the biomimetic simulation of biomaterials for bone tissue repair and engineering.     

Mechanisms of fluid-flow-induced matrix production in bone tissue engineering

Matrix production by tissue-engineered bone is enhanced when the growing tissue is subjected to mechanical forces and/or fluid flow in bioreactor culture. Cells deposit collagen and mineral, depending upon the mechanical loading that they receive. However, the molecular mechanisms of flow-induced signal transduction in bone are poorly understood. The hyaluronan (HA) glycocalyx has been proposed as a potential mediator of mechanical forces in bone. Using a parallel-plate flow chamber the effects of removal of HA on flow-induced collagen production and NF-kappaB activation in MLO-A5 osteoid osteocytes were investigated. Short periods of fluid flow significantly increased collagen production and induced translocation of the NF-kappaB subunit p65 to the cell's nuclei in 65 per cent of the cell population. Enzymatic removal of the HA coat and antibody blocking of CD44 (a transmembrane protein that binds to HA) eliminated the fluid-flow-induced increase in collagen production but had no effect on the translocation of p65. HA and CD44 appear to play roles in transducing the flow signals that modulate collagen production over long-term culture but not in the short-term flow-induced activation of NF-kappaB, implying that multiple signalling events are initiated from the commencement of flow. Understanding the mechanotransduction events that enable fluid flow to stimulate bone matrix production will allow the optimization of bioreactor design and flow profiles for bone tissue engineering.     

Effects of the sample preparation temperature on the nanostructure of compression moulded ultrahigh molecular weight polyethylene

In this study, the effects of the sample sectioning temperature on the surface nanostructure and mechanical response of compression moulded ultrahigh molecular weight polyethylene (UHMWPE) at a nanometer scale (nanomechanical properties) have been characterized. The primary focus of this work was to determine if the sample sectioning temperature significantly changed the nanostructure of UHMWPE, while the secondary focus was to characterize the effect on the mechanical response due to the changes in the sectioned surface nanostructure. The goals of this study were: (a) to investigate the potential possibility of creating surface artefacts by the sample preparation technique by sectioning at different temperatures relative to the published range of glass transition temperatures, Tg, for PE (-12, -80 and -25 degrees C); (b) to determine the possibility of molecular orientation induced by plastic deformation of the UHMWPE sample during the process of sample preparation; (c) to measure the relative difference in nanomechanical properties owing to evolution of different nanostructures as a function of sample sectioning temperature. Field emission scanning electron microscopy (FESEM), atomic force microscopy (AFM) and nanoindentation were used to demonstrate that the sectioning temperature caused a change in nanostructure of the compression moulded UHMWPE sectioned surface, explaining the change in mechanical response to indentation at a nanoscale. In this study, it was demonstrated that significant plastic deformation occurs when a shear stress is applied between the glass or diamond blade and the UHMWPE during sample preparation under ambient conditions at a temperature of 22 degrees C. These results also suggest that an optimum sample sectioning temperature should definitely be below the measured Tg of the polymer.     

Visualization of the nanoscale assembly of type I collagen on PLA by AFM

Collagen adsorption and the morphology of its assemblies at polymer surface play an important role in improving the biocompatibility of materials. In this study, the nanoscale organization of type I collagen on Polylactide (PLA) was observed directly by high‐resolution atomic force microscopy. The results show that the supramolecular structure of adsorbed collagen was affected by the concentration of collagen solution, appropriate pH and electrolyte composition of the buffer. On PLA substrate, network structures formed in high humidity atmosphere. In addition, collagen formed well‐oriented nano‐patterns at nearly neutral pH and appropriate electrolyte composition. Particularly, the typical 65 nm D‐periodicity of collagen fibers was observed in the presence of potassium ions. Our investigation provides useful insights into the regulation of collagen assembly by substrates and environmental conditions, which is important for understanding the mechanism of collagen adsorption and assembly on polymer surfaces. It also offers a potential way to create surfaces of bio‐functioned and nano‐patterned materials for biotechnological and biomedical applications. SCANNING 32: 104–111, 2010. © 2010 Wiley Periodicals, Inc.     

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an <i>in vivo</i> model

This article presents the construction of scaffolds composed of polylactic acid (PLA) with different concentrations of hydroxyapatite (HA) by electrospinning, which were superficially modified with polypyrrole (PPy/I) by plasma polymerization. A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days; bone cells differentiated from mesenchymal stem cells (MSCs) were used. The bone cell and scaffold structures were characterized by histological, immunohistochemical, and mechanical stress tests. Hematoxylin–eosin staining showed good tissue conformation. The immunohistochemical tests highlighted the presence of the main bone tissue proteins, such as collagen, osteocalcin, and osteopontin. The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties; however, the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I. The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support, allowing the formation of neotissues with good viability of cell growth.     

Fabrication and in vivo evaluation of Ti6Al4V implants with controlled porous structure and complex shape

Electron beam melting process was used to fabricate porous Ti6Al4V implants. The porous structure and surface topography of the implants were characterized by scanning electron microscopy (SEM) and digital microscopy (DM). The results showed that the pore size was around 600 and the porosity approximated to 57%. There was about±50 μm of undulation on implants surfaces. Standard implants and a custom implant coupled with porous sections were designed and fabricated to validate the versatility of the electron beam melting (EBM) technique. After coated with bone-like apatite, samples with fully porous structures were implanted into cranial defects in rabbits to investigate the in vivo performance. The animals were sacrificed at 8 and 12 weeks after implantation. Bone ingrowth into porous structure was examined by histological analysis. The histological sections indicated that a large amount of new bone formation was observed in porous structure. The newly formed bone grew from the calvarial margins toward the center of the bone defect and was in close contact with implant surfaces. The results of the study showed that the EBM produced Ti6Al4V implants with well-controlled porous structure, rough surface topography and bone-like apatite layer are beneficial for bone ingrowth and apposition.     

Dynamic three-dimensional visualization of collagen matrix remodeling and cytoskeletal organization in living corneal fibroblasts

The remodeling of extracellular matrices by cells plays a defining role in developmental morphogenesis and wound healing, as well as in tissue engineering. Three-dimensional (3-D) type I collagen matrices have been used extensively as an in vitro model for studying cell-induced matrix reorganization at the macroscopic level. However, few studies have directly assessed the dynamic process of 3-D matrix remodeling at the cellular and subcellular level. We recently developed an experimental model for investigating cell-matrix mechanical interactions by plating green fluorescen protein (GFP)-zyxin transfected cells inside fibrillar collagen matrices and performing high-magnification time-lapse differential interference microscopy (DIC) and wide-field fluorescent imaging. In this study, we extend this experimental model by performing four-dimensional (4-D) reflected light and fluorescent confocal imaging (using either visible light or multiphoton excitation) of living corneal fibroblasts transfected to express GFP-zyxin or GFP-alpha-actinin, 18 h after plating inside 3-D collagen matrices. Reflected light confocal imaging allowed detailed visualization of the cells and the fibrillar collagen surrounding them. By overlaying maximum intensity projections of reflected light and GFP-zyxin or GFP-alpha-actinin images and generating stereo pair reconstructions, 3-D interactions between focal adhesions and collagen fibrils in living cells could be visualized directly. Focal adhesions were generally oriented parallel to the direction of collagen fibril alignment in front of the cell. Killing the cells induced relaxation of transient cell-induced tension on the matrix; however, significant permanent remodeling always remained. Time-lapse 3-D imaging demonstrated an active response to the Rho-kinase inhibitor Y-27632, as indicated by cell elongation, extracellular matrix relaxation, and extension of pseudopodial processes. It is interesting that, at higher cell densities, groups of collagen fibrils were compacted and aligned into straps between neighboring cells. Overall, the continued development and application of this new approach should provide important insights into the basic underlying biochemical and biomechanical regulatory mechanisms controlling matrix remodeling by corneal fibroblasts.     

Quantification of the graphical details of collagen fibrils in transmission electron micrographs

A novel 2D image analysis technique is demonstrated. Using the digitized images of articular cartilage from transmission electron microscopy (TEM), this technique performs a localized 'vector' analysis at each region that is large enough to include several or tens of collagen fibrils but small enough to provide a fine resolution for the whole tissue. For each small and localized region, the morphology of the collagen fibrils can be characterized by three quantities essential to the nature of the tissue: the concentration of the fibrils, the overall orientation of the fibrils, and the anisotropy of the fibrils. This technique is capable of providing new insight to the existing technology by assigning quantitative attributes to the qualitative graphics. The assigned quantities are sensitive to the fine structure of the collagen matrix and meaningful in the architectural nature of the collagen matrix. These quantities could provide a critical linkage between the ultrastructure of the tissue and the macroscopic behaviours of the material. In addition, coarse-graining the microscopic resolution of EM without compromising the essential features of the tissue's structure provides a direct view of the tissue's morphology and permits direct correlations and comparisons among interdisciplinary techniques.     

Accumulation of in-vivo fatigue microdamage and its relation to biomechanical properties in ageing human cortical bone

Bone matrix accumulates microdamage in the form of microcracks as a result of everyday cyclic loading activities. In two very recent studies, which used conventional histological stains and light microscopy techniques, the amount of this in-vivo microdamage in the cortices of long bones has been shown to increase with age. These articles have suggested that in-vivo microcracks may have an effect on the material properties of the tissue. However, a precise quantitative relationship between the number of microcracks and the mechanical properties of these same bones has not been produced before, and in particular the way the microcracks may affect the stiffness, the strength or possibly the toughness of the tissue. This article presents an examination of the in-vivo microdamage in human bones by the use of laser scanning confocal microscopy, which offers better discrimination and allows examination of the cracks in-situ . Quantification of in-vivo fatigue microcracks was performed by counting the microcrack numerical density and surface density in specimens for which we have previously derived a full set of mechanical properties as a function of age. It is shown that bone microdamage relates more to the toughness (measured by three different measures) of ageing bone tissue than to its stiffness and strength. The result allows us (i) to re-evaluate the fragility of ageing human bone and put more emphasis on its energy-related resistance to fracture than perhaps on its stiffness or strength and also (ii) to understand more fully the causal relationship and interactions between microcracks and tissue toughness.     

Macro- and Nanotribological Properties of Graphite Tribofilms: Influence of the Sliding Interface

Macrotribological studies of microcrystalline graphite powder reveal a drastic decrease in the friction coefficient when the experiments are carried out in the presence of low-viscosity liquids. The friction reduction is attributed to the simultaneous presence of particles and liquid in the sliding contact, but the mechanisms involved remain unclear. In order to contribute to the understanding of liquid action in friction reduction mechanisms, nanoscale investigations of the tribofilms have been performed using lateral force microscopy. Attention is devoted to the nanostructure of the film surfaces and their nanofriction behavior using an atomic force microscope. The influence of the tip/sample interfaces on friction properties is investigated by using AFM tips constituted of different compounds (silicon, gold/chromium alloy, silicon nitride or carbon-covered AFM tip) and by performing the nanofriction tests in air or liquid environments. The results indicate that the friction reduction observed at macroscale is attributed neither to the lowering of the shear strength of the carbon/carbon interface in the presence of liquid nor to the nanostructure of the film surface. Collective liquid/particles effects inside the contact during sliding are probably involved.     

Photoplastic near-field optical probe with sub-100 nm aperture made by replication from a nanomould

Polymers have the ability to conform to surface contours down to a few nanometres. We studied the filling of transparent epoxy‐type EPON SU‐8 into nanoscale apertures made in a thin metal film as a new method for polymer/metal near‐field optical structures. Mould replica processes combining silicon micromachining with the photo‐curable SU‐8 offer great potential for low‐cost nanostructure fabrication. In addition to offering a route for mass production, the transparent pyramidal probes are expected to improve light transmission thanks to a wider geometry near the aperture. By combining silicon MEMS, mould geometry tuning by oxidation, anti‐adhesion coating by self‐assembled monolayer and mechanical release steps, we propose an advanced method for near‐field optical probe fabrication. The major improvement is the possibility to fabricate nanoscale apertures directly on wafer scale during the microfabrication process and not on free‐standing tips. Optical measurements were performed with the fabricated probes. The full width half maximum after a Gaussian fit of the intensity profile indicates a lateral optical resolution of ≈ 60 nm.     

Mechanical testing of recombinant human bone morphogenetic protein-7 regenerated bone in sheep mandibles

A new method was developed in this study for testing excised sheep mandibles as a cantilever. The method was used to determine the strength and stiffness of sheep hemi-mandibles including a 35 mm defect bridged by regenerated bone. Recombinant human bone morphogenetic protein-7 (rhBMP-7) in a bovine collagen type-I carrier was used for the bone regeneration. Initial tests on ten intact sheep mandibles confirmed that the strength, stiffness and area beneath the load-deformation curves of the right and left hemi-mandibles were not significantly different, confirming the validity of using the contra-lateral hemi-mandible as a control side. Complete bone regeneration occurred in six hemi-mandibles treated with rhBMP, but the quality and mechanical properties of the bone were very variable. The new bone in three samples contained fibrous tissue and was weaker and less stiff than the contra-lateral side (strength, 10-20 per cent; stiffness, 6-15 per cent). The other half had better-quality bone and was significantly stiffer and stronger (p < 0.05), with strength 45-63 per cent and stiffness 35-46 per cent of the contra-lateral side. Hemi-mandibles treated with collagen alone had no regenerated bone bridge suggesting that 35 mm is a critical-size bone defect.