A comparison of stereology, structural rigidity and a novel 3D failure surface analysis method in the assessment of torsional strength and stiffness in a mouse tibia fracture model

In attempting to develop non-invasive image based measures for the determination of the biomechanical integrity of healing fractures, traditional μCT based measurements have been limited. This study presents the development and evaluation of a tool for assessment of fracture callus mechanical properties through determination of the geometric characteristics of the fracture callus, specifically along the surface of failure identified during destructive mechanical testing. Fractures were created in tibias of ten male mice and subjected to μCT imaging and biomechanical torsion testing. Failure surface analysis, along with previously described image based measures was calculated using the μCT image data, and correlated with mechanical strength and stiffness. Three-dimensional measures along the surface of failure, specifically the surface area and torsional rigidity of bone, were shown to be significantly correlating with mechanical strength and stiffness. It was also shown that surface area of bone along the failure surface exhibits stronger correlations with both strength and stiffness than measures of average and minimum torsional rigidity of the entire callus. Failure surfaces observed in this study were generally oriented at 45° to the long axis of the bone, and were not contained exclusively within the callus. This work represents a proof of concept study, and shows the potential utility of failure surface analysis in the assessment of fracture callus stability.     

Bone grafts: a radiologic, histologic, and biomechanical model comparing autografts, allografts, and free vascularized bone grafts

We developed an experimental model to compare the efficacy of free vascularized bone grafts, conventional segmental autografts, matchstick autografts, and fresh segmental allografts in terms of their ability to reconstruct a 7-cm segmental diaphyseal defect created in the canine femur. Forty-five adult mongrel dogs were studied and followed for 6 to 12 months prior to sacrifice. Evaluation included radiologic assessment of graft incorporation and hypertrophy, histology, and biomechanical testing. These studies indicated that microsurgically revascularized autografts were superior to all other groups in terms of early incorporation, hypertrophy, and the highest mechanical strength to failure. Union of the bone graft to the recipient femur was achieved by 6 months in 25 of 26 autografts, and no difference in union rate was seen within the autograft group. However, only two of five allografts achieved bony union during this time interval. Arteriography, microangiography, fluorochrome, and histologic studies all supported the concept that microsurgically revascularized grafts, when successful, maintain their viability. However, the premise that all osteocytes survive in a successfully revascularized bone graft is open to question. While decalcified sections showed that all microsurgically revascularized grafts maintained normal viability in the central marrow and cancellous portions compared with the other three groups, the viability of cortical bone in the vascularized autografts was less clear. Undecalcified fluorochrome sections suggested that circulation was not preserved in all portions of the cortex. Revascularization of the nonvascularized autografts was complete at 3 months, while, in the avascular allografts, the process was not complete at 6 months.     

Sterilization with electron beam irradiation influences the biomechanical properties and the early remodeling of tendon allografts for reconstruction of the anterior cruciate ligament (ACL)

Although allografts for anterior cruciate ligament (ACL) replacement have shown advantages compared to autografts, their use is limited due to the risk of disease transmission and the limitations of available sterilization methods. Gamma sterilization has shown detrimental effects on graft properties at the high doses required for sufficient pathogen inactivation. In our previous in vitro study on human patellar tendon allografts, Electron beam (Ebeam) irradiation showed less detrimental effects compared to gamma sterilization (Hoburg et al. in Am J Sports Med 38(6):1134-1140, 2010). To investigate the biological healing and restoration of the mechanical properties of a 34?kGy Ebeam treated tendon allograft twenty-four sheep underwent ACL replacement with either a 34?kGy Ebeam treated allograft or a non-sterilized fresh frozen allograft. Biomechanical testing of stiffness, ultimate failure load and AP-laxity as well as histological analysis to investigate cell, vessel and myofibroblast-density were performed after 6 and 12?weeks. Native sheep ACL and hamstring tendons (HAT, each n?=?9) served as controls. The results of a previous study analyzing the remodeling of fresh frozen allografts (n?=?12) and autografts (Auto, n?=?18) with the same study design were also included in the analysis. Statistics were performed using Mann-Whitney U test followed by Bonferroni-Holm correction. Results showed significantly decreased biomechanical properties during the early remodeling period in Ebeam treated grafts and this was accompanied with an increased remodeling activity. There was no recovery of biomechanical function from 6 to 12?weeks in this group in contrast to the results observed in fresh frozen allografts and autografts. Therefore, high dose Ebeam irradiation investigated in this paper cannot be recommended for soft tissue allograft sterilization.     

Biomechanical Strength of Large Diaphyseal Deep-frozen Allografts

The aim of this paper is to study the biomechanical strength of deep-frozen allografts as they heal. Twenty-eight adult cats were used with the tibia as the experimental model site. Deep-frozen allografts stored at -80°C were used to reconstruct a large tibial defect (at least two-thirds of the diaphysis). An intra-medullary rod was used for fixation. The healing was studied by X-ray at observation periods of 4, 6, 8, 12, 16, 24 and 36 weeks. Post-transplantation biomechanical testing was performed using the Shimadzu Universal Testing Machine DCS series with a torsion test device of 50kg force metre. Parameters studied included maximum torque, torsional stiffness and energy of absorption. The transplanted grafts were compared to the mechanical properties of the internal controls of the normal opposite tibia of each cat. The results of the mechanical tests demonstrated that deep-frozen allografts did not regain normal strength. At nine months, only about 60% of normal torque strength and about 80% of normal torsional stiffness was achieved. Clinically, it is important to employ strong and rigid internal fixation using intra-medullary nailing rather than plating to allow for immediate mobilisation and reduce the rate of graft fracture.     

Non-Invasive Monitoring of Temporal and Spatial Blood Flow during Bone Graft Healing Using Diffuse Correlation Spectroscopy

Vascular infiltration and associated alterations in microvascular blood flow are critical for complete bone graft healing. Therefore, real-time, longitudinal measurement of blood flow has the potential to successfully predict graft healing outcomes. Herein, we non-invasively measure longitudinal blood flow changes in bone autografts and allografts using diffuse correlation spectroscopy in a murine femoral segmental defect model. Blood flow was measured at several positions proximal and distal to the graft site before implantation and every week post-implantation for a total of 9 weeks (autograft n = 7 and allograft n = 10). Measurements of the ipsilateral leg with the graft were compared with those of the intact contralateral control leg. Both autografts and allografts exhibited an initial increase in blood flow followed by a gradual return to baseline levels. Blood flow elevation lasted up to 2 weeks in autografts, but this duration varied from 2 to 6 weeks in allografts depending on the spatial location of the measurement. Intact contralateral control leg blood flow remained at baseline levels throughout the 9 weeks in the autograft group; however, in the allograft group, blood flow followed a similar trend to the graft leg. Blood flow difference between the graft and contralateral legs (ΔrBF), a parameter defined to estimate graft-specific changes, was elevated at 1–2 weeks for the autograft group, and at 2–4 weeks for the allograft group at the proximal and the central locations. However, distal to the graft, the allograft group exhibited significantly greater ΔrBF than the autograft group at 3 weeks post-surgery (p < 0.05). These spatial and temporal differences in blood flow supports established trends of delayed healing in allografts versus autografts.     

Clinicopathologic studies on human epithelial autografts and allografts.

We compared the survival of cultured epithelial allografts and epithelial autografts applied to donor sites for split-thickness skin grafts. Before grafting, cultured epithelium was devoid of Langerhans cells (LCs) or lymphoid cells by immunohistochemical and electron microscopic examinations. The autografts attached to the wounds permanently, without any clinical evidence of rejection. In contrast, allografts, which were mismatched for MHC and blood-type antigens, appeared to adhere firmly only until day 7. By the second week, signs of graft rejection were apparent: The graft changed color, and the underlying dermis underwent "microerosion" and denudation. By the third week, the area formerly occupied by the allograft had the same coloration as ungrafted wounds and apparently had undergone reepithelialization by the host. Immunohistochemical and ultrastructural studies clearly demonstrated that host Langerhans-like cells (without Birbeck granules) appeared in both autografts and allografts. However, these cells were numerous and distributed widely throughout allografts, whereas they were scarce and confined to the basal layer of autografts. Typical Langerhans cells (containing Birbeck granules) were present in the prickle-cell layer of autografts by day 7. The present study strongly indicates that allografts of cultured epithelium are rejected. Furthermore, given the known ability of Langerhans-like cells to function as accessory cells in T-cell activation, our results point to a role for host Langerhans-like cells in immunologically mediated rejection of the epithelial allografts.     

The differentiation of bone marrow cells to functional T lymphocytes following implantation of thymus grafts and thymic stroma in nude and AT×BM mice

Cardiac allografts were used to compare the immunologic capacity of nude mice and adult, thymectomized, lethally irradiated, bone marrow-reconstituted (AT × BM) mice. Neither nude nor AT × BM mice were able to reject cardiac allografts of any party. However, both rejected grafts of any party following implantation of neonatal thymus or thymus from 3-week-old syngeneic mice. Irradiated syngeneic thymus grafts (800 R) were equally effective in restoring host responsiveness against allografts. In contrast, allogeneic thymus grafts restored the capacity to reject second-party heart grafts only in AT × BM mice. Second-party grafts persisted indefinitely when placed on nude mice implanted with an allogeneic, unirradiated thymus graft. Third-party grafts transplanted 17 weeks after reconstitution, however, were rejected. Irradiated nude mice given normal littermate bone marrow and simultaneously grafted with second-party thymus and heart allografts also failed to reject their second-party heart grafts. The difference in ultimate capacity to respond between AT × BM and nude mice suggests that a maturational defect exists in the nude mouse enviroment which impedes development of precursor T lymphocytes.     

Traumatic brain injury and bone healing: radiographic and biomechanical analyses of bone formation and stability in a combined murine trauma model

Introduction:The combination of traumatic brain injury (TBI) and long-bone fractures has previously been reported to lead to exuberant callus formation. The aim of this experimental study was to radiographically and biomechanically study the effect of TBI on bone healing in a mouse model.Materials and methods:138 female C57/Black6N mice were assigned to four groups (fracture (Fx) / TBI / combined trauma (Fx/TBI) / controls). Femoral osteotomy and TBI served as variables: osteotomies were stabilized with external fixators, TBI was induced with controlled cortical impact injury. During an observation period of four weeks, in vivo micro-CT scans of femora were performed on a weekly basis. Biomechanical testing of femora was performed ex vivo.Results:The combined-trauma group showed increased bone volume, higher mineral density, and a higher rate of gap bridging compared to the fracture group. The combined-trauma group showed increased torsional strength at four weeks.Discussion:TBI results in an increased formation of callus and mineral density compared to normal bone healing in mice. This fact combined with a tendency towards accelerated gap bridging leads to increased torsional strength. The present study underscores the empirical clinical evidence that TBI stimulates bone healing. Identification of underlying pathways could lead to new strategies for bone-stimulating approaches in fracture care.     

Solvent-dehydrated calvarial allografts in craniofacial surgery

Craniofacial surgery almost always requires the use of bone grafting. Although autografts are the standard procedure for bone grafting, it is sometimes not possible to harvest bone, and autografts have particular risks. The use of allograft bone provides a reasonable alternative to meet the need for graft material. Solvent dehydration is a multistage procedure in which human cadaveric bone is processed by osmotic exchange baths and gamma sterilization. This processing avoids the risk of infection transmission, decreases antigenicity, and does not weaken the mechanical properties of the bone. Solvent-dehydrated, gamma-irradiated human calvarial bone allografts were used for reconstruction of craniofacial deformities in 24 patients between 1988 and 2002. Resorption of the allografts and results of the surgical intervention were evaluated with plain radiographs and three-dimensional computed tomography 12 months after surgery, in 21 patients. Serologic tests for human immunodeficiency virus-1 antibody, hepatitis B surface antigen, and hepatitis C antigen were also performed. Biopsy specimens were taken from the allografts. Average follow-up in this group was 30 months (range, 8 to 60 months), and results of serologic tests were negative in all patients. Seventy-one percent of the patients (15 of 21) showed no resorption, with partial and complete allograft fusion. One patient had nearly total graft loss and the remaining five patients had 10 to 25 percent graft resorption. Rigid fixation of the allograft, contact with the dura and periosteum, and prevention of dead spaces around the allograft are the most important factors in achieving a satisfactory result. In solvent-dehydrated bone allografts, sterilization and antigenic tissue cleaning are achieved after several steps with a minimal dose of radiation. The result is a nonantigenic, sterile mechanical scaffold that can tolerate external forces. Although autografts are the standard in craniofacial surgery, solvent-dehydrated calvarial bone allografts produced successful results in selected cases.     

Is callus calcium content an indicator of the mechanical strength of healing fractures? An experimental study in rat metatarsals

Changes in the mechanical properties and the calcium content of healing fracture callus were followed, using rat metatarsals. By 24 weeks post-fracture the mean ultimate tensile stress and elastic modulus were still less than half that of the contralateral unfractured bone, whereas the mean torsional modulus had almost reached that of the unfractured bone. The calcium content of the callus formed immediately between the fractured ends of the bone showed changes which coincided with the increases in mechanical strength and the moduli, thus measurement of callus calcium content would enable the prediction of the strength of a healing fracture.     

Non-invasive assessment of failure torque in rat bones with simulated lytic lesions using computed tomography based structural rigidity analysis

This study applies CT-based structural rigidity analysis (CTRA) to assess failure torque of rat femurs with simulated lytic defects at different locations (proximal and distal femur) and diameters (25% and 50% of the cross-section at the site), and compared the results to those obtained from mechanical testing. Moreover, it aims to compare the correlation coefficients between CTRA-based failure torque and DXA-based aBMD versus actual failure torque. Twenty rats were randomly assigned to four equal groups of different simulated lesions based on size and location. Femurs from each animal underwent micro-computed tomography to assess three-dimensional micro-structural data, torsional rigidity using structural rigidity analysis and dual energy X-ray absorptiometry to assess bone mineral density. Following imaging, all specimens were subjected to torsion. Failure torque predicted from CT-derived structural rigidity measurements was better correlated with mechanically derived failure torque [R(2)=0.85] than was aBMD from DXA [R(2)=0.32]. In summary, the results of this study suggest that computed tomography based structural rigidity analysis can be used to accurately and quantitatively measure the mechanical failure torque of bones with osteolytic lesions in an experimental rat model. Structural rigidity analysis can provide more accurate predictions on maximal torque to mechanical failure than dual energy X-ray absorptiometry based on bone mineral density.     

Virtual structural analysis of tibial fracture healing from low-dose clinical CT scans

Quantitative assessment of bone fracture healing remains a significant challenge in orthopaedic trauma research. Accordingly, we developed a new technique for assessing bone healing using virtual mechano-structural analysis of computed tomography (CT) scans. CT scans from 19 fractured human tibiae at 12 weeks after surgery were segmented and prepared for finite element analysis (FEA). Boundary conditions were applied to the models to simulate a torsion test that is commonly used to access the structural integrity of long bones in animal models of fracture healing. The output of each model was the virtual torsional rigidity (VTR) of the healing zone, normalized to the torsional rigidity of each patient’s virtually reconstructed tibia. This provided a structural measure to track the percentage of healing each patient had undergone. Callus morphometric measurements were also collected from the CT scans. Results showed that at 12 weeks post-op, more than 75% of patients achieved a normalized VTR (torsional rigidity relative to uninjured bone) of 85% or above. The predicted intact torsional rigidities compared well with published cadaveric data. Across all patients, callus volume and density were weakly and non-significantly correlated with normalized VTR and time to clinical union. Conversely, normalized VTR was significantly correlated with time to union (R² = 0.383, p = 0.005). This suggests that fracture scoring methods based on the visual appearance of callus may not accurately predict mechanical integrity. The image-based structural analysis presented here may be a useful technique for assessment of bone healing in orthopaedic trauma research.     

Quantitative,structural, and image-based mechanical analysis of nonunion fracture repaired by genetically engineered mesenchymal stem cells

Stem cell-mediated gene therapy for fracture repair, utilizes genetically engineered mesenchymal stem cells (MSCs) for the induction of bone growth and is considered a promising approach in skeletal tissue regeneration. Previous studies have shown that murine nonunion fractures can be repaired by implanting MSCs over-expressing recombinant human bone morphogenetic protein-2 (rhBMP-2). Nanoindentation studies of bone tissue induced by MSCs in a radius fracture site indicated similar elastic modulus compared to intact murine bone, eight weeks post-treatment. In the present study we sought to investigate temporal changes in microarchitecture and biomechanical properties of repaired murine radius bones, following the implantation of MSCs. High-resolution micro-computed tomography (micro-CT) was performed 10 and 35 weeks post MSC implantation, followed by micro-finite element (micro-FE) analysis. The results have shown that the regenerated bone tissue remodels over time, as indicated by a significant decrease in bone volume, total volume, and connectivity density combined with an increase in mineral density. In addition, the axial stiffness of limbs repaired with MSCs was 2–1.5 times higher compared to the contralateral intact limbs, at 10 and 35 weeks post-treatment. These results could be attributed to the fusion that occurred in between the ulna and radius bones. In conclusion, although MSCs induce bone formation, which exceeds the fracture site, significant remodeling of the repair callus occurs over time. In addition, limbs treated with an MSC graft demonstrated superior biomechanical properties, which could indicate the clinical benefit of future MSC application in nonunion fracture repair.     

Mineral and matrix contributions to rigidity in fracture healing

The purpose of this study was to investigate the relationships among selected properties of fracture callus: bending rigidity, tissue density, mineral density, matrix density and mineral-to-matrix ratio. The experimental model was an osteotomized canine radius in which the development of the fracture callus was modified by electrical stimulation with various levels of direct current. This resulted in a range of values for the selected properties of the callus, determined post mortem at 7 weeks after osteotomy. We found that the rigidity (R) of the bone-callus combination obeyed relationships of the form R = axb, where x is the tissue density, mineral density, matrix density or the mineral-to-matrix ratio of the repair tissue. These are analogous to power-law relationships found in studies of compact and cancellous bone. The results suggest that fracture callus at 7 weeks after osteotomy in canine radius behaves more like immature compact bone than cancellous bone in its mechanical and physicochemical properties. The present study demonstrates the feasibility of developing non-invasive in vivo densitometric methods to monitor fracture healing, since models may be developed that can predict mechanical properties from densitometric data. Further studies are needed to develop a refined model based on experimental data on the mechanical and physicochemical properties and microstructure of fracture callus at different stages of healing.     

The Use of Irradiated Allografts in Reconstruction of Tumor Defects – the Tata Memorial Hospital Experience

A Tissue Bank is a valuable adjunct to tumour management. In bone tumours, the defects produced by ablative surgery can be reconstructed using banked tissue, thereby obviating the donor site morbidity associated with autografts. Allografts are especially useful in large defects or in children where the quantity of available autograft is limited. The use of bone allografts in India has been limited by the availability of good quality, affordable grafts. In this article we share our experience with the use of indigenously produced allografts in limb salvage, as bone graft expanders and as struts. Lyophilised, irradiated bone allografts were morcellised and used in 32 patients. In 21 of these patients the allograft was used in contained cavities. Complete incorporation of the graft was seen between 6-9 months in all the 25 cases available for follow-up. In 4 patients the allograft was layered onto autograft. The allograft incorporated with the host bone in only one of these patients.Struts were used in 9 cases (3 cases complete intercalary segmental defect, 3 cases of hemicortical defects, 2 cases of allograft-prosthesis composite around the hip, 1 case an iliac-crest block was used to stop bleeding from an anterior sacral defect). Of these, no incorporation of the full segment struts was observed in 2 patients who were on chemotherapy and radiotherapy. The sacral defect case was lost to follow-up. All the other struts incorporated with the host bone within 6-9 months. In 5 cases there was sterile postoperative drainage. Overall infection was observed in 4 patients (10%). In one the graft was removed, another settled uneventfully with subsequent incorporation of graft, and two have a persisting sinus but good incorporation. Since radiation and lyophilisation are known to affect the material properties of bone, the grafts were rehydrated in saline for 30 minutes prior to transplantation. Autogenous marrow or autograft was used to provide osteoinductive properties. In selected cases the lyophilised, irradiated bone allografts proved to be clinically useful in the reconstruction of large tumour defects.     

Earthworm leukocyte interactions during early stages of graft rejection

The earliest phase of graft rejection in earthworms, the recognition of foreign tissue antigens, has been subjected to analysis by confronting host leukocytes with foreign erythrocytes. Only rabbit and rat erythrocytes significantly prevented healing of allografts when grafts were transplanted and erythrocytes injected simultaneously. In contrast, autografts and allografts transplanted on worms injected 1 or 2 days before grafting were never affected. Since earthworms readily produce higher titers of erythrocyte agglutinins at 24 h postinjection than at later times, we propose a hypothetical scheme of earthworm leukocyte interactions that may occur during the early phases of graft healing and of agglutinin synthesis.     

Reconstructing Tumour Defects: Lyophilised, Irradiated Bone Allografts

Tumour excision leaves behind large defects. Allografts provide an excellent alternative to autografts without donor site morbidity and are especially useful in large defects or in children where the quantity of available autograft is limited. In this paper we discuss our experience with indigenously procured and processed lyophilised, irradiated bone allografts. Bone allografts were used in 41 patients. They were used morsellised and used in 32 cases. Of these, 25 cases were available for follow-up. These included 21 patients in whom the allograft was used in contained cavities. Complete incorporation of the graft was seen between 6 and 9 months in all these 21 patients. In 4 patients the allograft was layered onto autograft. In only one of these the allograft incorporated with the host bone. Struts were used in 9 cases (3 cases complete intercalary segmental defect, 3 cases of hemicortical defects, 2 cases of allograft-prosthesis composite around the hip, in 1 case an iliac-crest block was used to stop bleeding from an anterior sacral defect). Of these, 2 full segment struts showed no incorporation. Both these patients were on chemotherapy and radiotherapy. There was no follow-up in sacral defect case. All the other struts incorporated with the host bone within 6-9 months.In 5 cases there was sterile postoperative drainage. All these cases went on to uneventful. Deep infection was observed in 4 patients (10%). In one, the graft was removed, another settled uneventfully with subsequent incorporation of graft, and two have a persisting sinus but good incorporation.To restore part of the strength of the struts it was necessary to hydrate them for 30 min prior to use. Autogenous marrow or autograft was used to provide osteoinductive properties.Conclusion. In selected cases the lyophilised, irradiated bone allografts proved to be very useful in reconstruction of large tumour defects.     

Albumin-coated structural lyophilized bone allografts: a clinical report of 10 cases

Bone replacement and the use of bone supplementary biological substances have become widespread in clinical practice. Although autografts have excellent properties, their limited availability, difficulties with shaping and donor site morbidity have made allografts a viable and increasingly preferred alternative. The main drawback of allografts is that the preparation destroys osteogenic cells and results in denaturation of osteoinductive proteins. Serum albumin is a well-known constituent of stem cell culture media and we found that lyophilizing albumin onto bone allografts markedly improves stem-cell attachment and bone healing in animal models thus replacing some of the osteoinductive potential. As a first step in the clinical introduction of albumin coated grafts, we aimed to test surgical handling and early incorporation in aseptic revision arthroplasty in humans. We selected patients who needed large structural allografts and the current operation was the last attempt at preserving a moving joint. In a series of 10 cases of hip and knee revision surgery we did not experience any drawbacks of the albumin-coated grafts during handling and implantation. Twelve months radiographic and SPECT-CT follow-up showed that the graft was well received by the host and active remodelling was observed. The lack of graft-related complications and the good 1-year results indicate that controlled trials may be initiated in more common bone grafting indications where long-term effectiveness can be evaluated.     

Systemic Treatment with Vanadium Absorbed by Coprinus comatus Promotes Femoral Fracture Healing in Streptozotocin-Diabetic Rats

The purpose of this study was to analyze the impact of vanadium absorbed by Coprinus comatus (VACC) on fracture healing in streptozotocin-diabetic rats. Forty-five male Wistar rats used were divided into three groups: normal rats (control), diabetic rats, and diabetic rats treated with VACC. A standardized fracture-healing model with a stable plate fixation was established for the rat femoral fracture. After a 4-week stable fixation, callus quality was assessed by microcomputerized tomography and histological and biomechanical examinations. In addition, bone samples were obtained to evaluate the content of mineral substances in bones. Compared with the diabetic group, vanadium treatment significantly increased bone mineral content and biomechanical strength and improved microstructural properties of the callus. The ultimate load was increased by 29.1 % (P?<?0.05), and the total bone volume of callus enhanced by 11.2 % (P?<?0.05) at 4 weeks post fracture. Vanadium also promoted callus bone formation, which caused a 35.5 % increase in the total area of callus. However, VACC did not accelerate the fracture repair process in histological analysis. In conclusion, the current study suggests that systemic treatment with vanadium could promote fracture healing in streptozotocin-diabetic rats.     

Experimental study in rat metatarsals to determine whether callus vascularity is an indicator of the mechanical strength of healing fractures

Changes in the mechanical properties and percentage area of blood vessels of healing fracture callus were followed using rat metatarsals. By 24 weeks after fracture the mean ultimate tensile stress and elastic modulus were still less than half that of the contralateral unfractured bone, whereas the mean torsional modulus had almost reached that of the unfractured bone. The percentage area of blood vessels declined from five days post-fracture and showed no changes which coincided with the increases in mechanical strength or moduli. We conclude that studies of vascularity would not justify a prediction of the strength of a healing fracture.