Extracellular Calcium Ion Concentration Regulates Chondrocyte Elastic Modulus and Adhesion Behavior.

Extracellular calcium ion concentration levels increase in human osteoarthritic (OA) joints and contribute to OA pathogenesis. Given the fact that OA is a mechanical problem, the effect of the extracellular calcium level ([Ca2+]) on the mechanical behavior of primary human OA chondrocytes remains to be elucidated. Here, we measured the elastic modulus and cell-ECM adhesion forces of human primary chondrocytes with atomic force microscopy (AFM) at different extracellular calcium ion concentration ([Ca2+]) levels. With the [Ca2+] level increasing from the normal baseline level, the elastic modulus of chondrocytes showed a trend of an increase and a subsequent decrease at the level of [Ca2+], reaching 2.75 mM. The maximum increment of the elastic modulus of chondrocytes is a 37% increase at the peak point. The maximum unbinding force of cell-ECM adhesion increased by up to 72% at the peak point relative to the baseline level. qPCR and immunofluorescence also indicated that dose-dependent changes in the expression of myosin and integrin ß1 due to the elevated [Ca2+] may be responsible for the variations in cell stiffness and cell-ECM adhesion. Scratch assay showed that the chondrocyte migration ability was modulated by cell stiffness and cell-ECM adhesion: as chondrocyte's elastic modulus and cell-ECM adhesion force increased, the migration speed of chondrocytes decreased. Taken together, our results showed that [Ca2+] could regulate chondrocytes stiffness and cell-ECM adhesion, and consequently, influence cell migration, which is critical in cartilage repair.

Is diffusion anisotropy a biomarker for disease severity and surgical prognosis of cervical spondylotic myelopathy?

PURPOSE: To explore the value of diffusion-tensor (DT) imaging in addressing the severity of cervical spondylotic myelopathy (CSM) and predicting the outcome of surgical treatment. MATERIALS AND METHODS: From July 2009 to May 2012, 65 volunteers were recruited for this institutional review board-approved study, and all gave informed consent; 20 volunteers were healthy subjects (age range, 41-62 years), and 45 were patients with CSM (age range, 43-86 years). Anatomic and DT 3.0-T magnetic resonance images were obtained. Surgical decompression was performed in 22 patients with CSM, and patients were followed up for 6 months to 2 years. The clinical severity of myelopathy and postoperative recovery were assessed by using the modified Japanese Orthopaedic Association (mJOA) score. A recovery ratio (comparison of postoperative with preoperative mJOA score) of more than 50% indicated a good clinical outcome of surgery. DT findings, patient age, T2 high signal intensity (HSI), and somatosensory evoked potential (SEP) were analyzed by using a logistic regression model to predict the surgical outcome of patients with CSM. RESULTS: A significant difference in cervical cord mean fractional anisotropy (FA) was found between healthy subjects and patients with CSM (0.65 ± 0.05 [standard deviation] vs 0.52 ± 0.13, P < .001). FA values were significantly correlated with the severity of neurologic dysfunction indicated by mJOA score (r(2) = 0.327, P = .016). Logistic regression analysis showed that mean FA (P = .030) and FA at the C2 vertebra (P = .035) enabled prediction of good surgical outcome; however, preoperative mJOA (P = .927), T2 HSI (P = .176), SEP amplitude (P = .154), and latency (P = .260) did not. CONCLUSION: FA is a biomarker for the severity of myelopathy and for subsequent surgical outcome.

Irradiation induces bone injury by damaging bone marrow microenvironment for stem cells.

Radiation therapy can result in bone injury with the development of fractures and often can lead to delayed and nonunion of bone. There is no prevention or treatment for irradiation-induced bone injury. We irradiated the distal half of the mouse left femur to study the mechanism of irradiation-induced bone injury and found that no mesenchymal stem cells (MSCs) were detected in irradiated distal femora or nonirradiated proximal femora. The MSCs in the circulation doubled at 1 week and increased fourfold after 4 wk of irradiation. The number of MSCs in the proximal femur quickly recovered, but no recovery was observed in the distal femur. The levels of free radicals were increased threefold at 1 wk and remained at this high level for 4 wk in distal femora, whereas the levels were increased at 1 wk and returned to the basal level at 4 wk in nonirradiated proximal femur. Free radicals diffuse ipsilaterally to the proximal femur through bone medullary canal. The blood vessels in the distal femora were destroyed in angiographic images, but not in the proximal femora. The osteoclasts and osteoblasts were decreased in the distal femora after irradiation, but no changes were observed in the proximal femora. The total bone volumes were not affected in proximal and distal femora. Our data indicate that irradiation produces free radicals that adversely affect the survival of MSCs in both distal and proximal femora. Irradiation injury to the vasculatures and the microenvironment affect the niches for stem cells during the recovery period.

Effect of tibial drill-guide angle on the mechanical environment at bone tunnel aperture after anatomic single-bundle anterior cruciate ligament reconstruction.

PURPOSE: The tibial drill-guide angle in anterior cruciate ligament (ACL) reconstruction influences the tunnel placement and graft-tunnel force, and is potentially associated with post-operative tunnel widening. This study aimed to examine the effect of the drill-guide angle on the stress redistribution at the tibial tunnel aperture after anatomic single-bundle ACL reconstruction. METHODS: A validated finite element model of human knee joint was used. The tibial tunnel with drill-guide angle ranging from 30° to 75° was investigated. The post-operative stress redistribution in tibia under the compressive, valgus, rotational and complex loadings was analysed. RESULTS: Compressive loading played a leading role on the stress redistribution at intra-articular tibial tunnel aperture. After ACL reconstruction, stress concentration occurred in the anterior and posterior regions of tunnel aperture while stress reduction occurred in the lateral and posteromedial regions under the compressive loading. Stress redistribution was partially alleviated by using the drill-guide angle ranging from 55° to 65°. CONCLUSIONS: The present study quantified the effect of bone tunnel drill-guide angle on the post-operative stress redistribution. This phenomenon potentially contributed to tunnel widening. A tunnel drill-guide angle ranging from 55° to 65° was proposed based on the biomechanical rationale. It could serve as a helpful surgical guide for ACL reconstruction.

Mechanistic links between systemic hypertension and open angle glaucoma.

Systemic hypertension or hypertension is a very common chronic age-related disease worldwide. It is typically characterised by a sustained elevation of blood pressure, particularly when the systolic blood pressure and/or diastolic blood pressure are of more than 140 mmHg and 90 mmHg, respectively. If hypertension is not well controlled, it may lead to an increased risk of stroke and heart attack. It has been shown that hypertension is linked to various ocular diseases, including cataract, diabetic retinopathy, age-related macular degeneration, and glaucoma. Glaucoma is the leading cause of irreversible blindness worldwide. Primary open angle glaucoma is the most common form of the disease and is usually characterised by an increase in intraocular pressure. This condition, together with normal tension glaucoma, constitutes open angle glaucoma. Systemic hypertension has been identified as a risk factor for open angle glaucoma. It is speculated that blood pressure is involved in the pathogenesis of open angle glaucoma by altering intraocular pressure or ocular blood flow, or both. Recent evidence has shown that both extremely high and low blood pressure are associated with increased risk of open angle glaucoma. Additional pathogenic mechanisms, including increased inflammation likely to be involved in the development and progression of these two diseases, are discussed.

Entropy-based analysis for diffusion anisotropy mapping of healthy and myelopathic spinal cord.

The present study utilized diffusion MR imaging and fractional anisotropy (FA) mapping to delineate the microstructure of spinal cord. The concept of Shannon entropy was introduced to analyze the complex microstructure of healthy and injured spinal cords based on FA map. A total of 30 volunteers were recruited in this study with informed consent, including 13 healthy adult subjects (group A, 25±3 years), 12 healthy elderly subjects (group B, 53±7 years) and 5 cervical spondylotic myelopathy (CSM) patients (group C, 53±15 years). Diffusion MRI images of cervical spinal cord were taken using pulsed gradient spin-echo-echo-planar imaging (SE-EPI) sequence with a 3T MR system. The region of interest was defined to cover the spinal cord in FA maps. The Shannon entropy of FA values of voxels in the cord was calculated as well as the average FA values. The significant differences were determined among three groups using one-way ANOVA and post-hoc test. As compared with adult and elderly healthy subjects, the entropy of whole spinal cord was significantly lower in CSM patients (group A: 6.07±0.18; B: 6.01±0.23; C: 5.32±0.44; p<0.05). Whereas there were no significant difference in FA values among groups (group A: 0.62±0.08; B: 0.64±0.09; C: 0.64±0.12). In CSM patients, there was a loss of architectural structural complexity in the cervical spinal cord tissue as noted by the lower Shannon entropy value. It indicated the potential application of entropy-based analysis for the diagnosis of the severity of chronic compressive spinal cord injuries, i.e. CSM.

Orientation entropy analysis of diffusion tensor in healthy and myelopathic spinal cord.

The majority of nerve fibers in the spinal cord run longitudinally, playing an important role in connecting the brain to the peripheral nerves. There is a growing interest in applying diffusion tensor imaging (DTI) to the evaluation of spinal cord microarchitecture. The current study sought to compare the organization of longitudinal nerve fibers between healthy and myelopathic spinal cords using entropy-based analysis of principal eigenvector mapping. A total of 22 subjects were recruited, including 14 healthy subjects, seven cervical myelopathy (CM) patients with single-level compression, and one patient suffering from multi-level compression. Diffusion tensor magnetic resonance (MR) images of the cervical spinal cord were obtained using a pulsed gradient, spin-echo echo-planar imaging (SE-EPI) sequence with a 3T MR system. Regions of interest (ROIs) were drawn manually to cover the spinal cord, and Shannon entropy was calculated in principal eigenvector maps. The results revealed no significant differences in orientation entropy values along the whole length of cervical spinal cord in healthy subjects (C2-3: 0.73±0.05; C3-4: 0.71±0.07; C4-5: 0.72±0.048; C5-6: 0.71±0.07; C6-7: 0.72±0.07). In contrast, orientation entropy values in myelopathic cord were significantly higher at the compression site (0.91±0.03), and the adjacent levels (above: 0.85±0.03; below: 0.83±0.05). This study provides a novel approach to analyze the orientation information in diffusion MR images of healthy and diseased spinal cord. These results indicate that orientation entropy can be applied to determine the contribution of each compression level to the overall disorganization of principal nerve tracts of myelopathic spinal cord in cases with multi-level compression.

Grafted tendon healing in tibial tunnel is inferior to healing in femoral tunnel after anterior cruciate ligament reconstruction: a histomorphometric study in rabbits.

PURPOSE: This study aimed to test whether graft healing in the tibial tunnel was inferior to that in the femoral tunnel after anterior cruciate ligament (ACL) reconstruction in rabbits. METHODS: Surgical reconstruction by use of the digital extensor tendon in the bone tunnel was performed in 18 rabbits. The rabbits were killed at weeks 2, 6, and 12 postoperatively, with 6 at each time point, for histologic examination. RESULTS: The transiently formed cartilaginous interface was gradually mineralized during re-establishment of direct tendon-to-bone integration, which was observed significantly less in the tibial tunnel than in the femoral tunnel (P < .05). The cell density of the graft was significantly lower in the tibial tunnel than that in the femoral tunnel at weeks 2 and 6 postoperatively (P < .05 for both). An increase in the immature type III collagen content was accompanied by a decrease in graft collagen fiber organization, with healing over time in both the femoral and tibial tunnels. The collagen fiber organization of the graft was significantly poorer in the tibial tunnel than that in the femoral tunnel at week 12 after surgery (P < .05). CONCLUSIONS: Grafted tendon healing in the tibial tunnel was inferior to that in the femoral tunnel at the tendon-to-bone interface and with regard to the grafted tendon within the bone tunnel after ACL reconstruction in rabbits. CLINICAL RELEVANCE: Future biopsy study is desirable to test whether this observation was valid clinically, which might provide a scientific basis for therapeutic targets to improve the outcome of ACL surgery.

Less Vertebral Bone Mass after Treatment with Macitentan in Mice: A Pilot Study.

PURPOSE: Blood vessels and skeleton interact together. Endothelin-1 is a potent vasoconstrictor and also has an effect on bone metabolism. The dual antagonist to both endothelin-1 type A and B receptors, Macitentan, has been approved for clinical management of pulmonary arterial hypertension while little is known about the secondary effect of the drug on spine. We aimed to answer how vertebral bone mass responded to Macitentan treatment in mice. METHODS: Sixteen male balb/c mice at 6 months were randomly assigned into 2 groups. Vehicle and Macitentan were administrated via intraperitoneal injection to Control group and Treatment group, respectively, for 4 months. At sacrifice, plasma endothelin-1 was evaluated with ELISA and vertebral bone mass was evaluated with Microcomputed Tomography and histological analysis. RESULTS: We found higher plasma endothelin-1 level (p<0.01) and less vertebral bone mass (p<0.05) in Treatment group compared to controls. Moreover, less osteoblasts and more osteoclasts were observed in the vertebral trabecular bone in the Treatment group compared to controls, by immunohistochemistry of the cell-specific markers. CONCLUSIONS: Treatment with Macitentan is associated with significant lower vertebral bone mass and therefore the secondary effect of dual antagonists to endothelin-1 receptors on the skeleton should be monitored and investigated in clinical practice. Both osteoblasts and osteoclasts may be involved while the molecular mechanism needs to be further explored.

High-Frequency Ultrasound Imaging of Tidemark In Vitro in Advanced Knee Osteoarthritis.

High-frequency ultrasound imaging has been widely adopted for assessment of the degenerative changes of articular cartilage in osteoarthritis (OA). Yet, there are few reports on investigating its capability to evaluate subchondral bone. Here, we employed high-frequency ultrasound imaging (25 MHz) to examine in vitro the tidemark in cylindrical osteochondral disks (n = 33) harvested from advanced OA knees of humans. We found good correspondence in morphology observed by ultrasound imaging and micro-computed tomography. Ultrasound roughness index (URI) of tidemark was derived from the raw radiofrequency signals to compare with bone quality factors, including bone volume fraction (BV/TV) and bone mineral density (BMD) measured by micro-computed tomography, using the Spearman correlation (ρ). URI of the tidemark was negatively associated with the subchondral plate BV/TV (ρ = -0.73, p <0.001), BMD (ρ = -0.40, p = 0.020), as well as the underneath trabecular bone BV/TV (ρ = -0.39, p = 0.025) and BMD (ρ = -0.43, p = 0.012). In conclusion, this preliminary study demonstrated that morphology measured by high-frequency ultrasound imaging could reflect the quality of the subchondral bone. High-frequency ultrasound is a promising imaging tool to evaluate the changes of the subchondral bone in addition to those of the overlying cartilage in OA.

Serum Osteocalcin and Testosterone Concentrations in Adult Males with or without Primary Osteoporosis: A Meta-Analysis.

Osteocalcin (Ocn) and testosterone play important roles in male skeleton. However, the concentrations of serum osteocalcin and testosterone have never been systematically compared between populations with and without primary male osteoporosis, a common skeletal disorder in adult males. We searched the PubMed, Embase, and Cochrane Library for relevant studies. A meta-analysis was performed to compare the serum osteocalcin and testosterone concentrations between primary osteoporotic males and age-matched nonosteoporotic (non-OP) males. Five case-control studies with 300 adult males were included. We found no significant difference between cases and controls in serum total osteocalcin (TOcn) [95% confidence interval (CI): -1.25, 1.31; p = 0.96] and total testosterone (TT) concentrations [95% CI: -0.88, 4.22; p = 0.20]. The level of evidence of this carefully performed meta-analysis is 3a according to Oxford (UK) CEBM Levels of Evidence. Future well-designed studies with larger sample size and better standardization of Ocn assay are awaited to confirm and update our current findings.

Subchondral bone proteomics in osteoarthritis: Current status and perspectives.

Osteoarthritis (OA) is the most common degenerative joint disorder. OA was conceived as a "wear and tear" problem of articular cartilage, yet there is a lack of treatment options to delay or rescue articular cartilage degeneration once it is established. Actually, the degradation of articular cartilage is related to a complex network of biochemical pathways involving the diffusion of catabolic factors within and between different joint tissues and particularly bone and cartilage. Advanced proteomics technology provides a powerful tool to allow us to build up a library of such factors. Factors that govern the bone-cartilage interplay could be the candidate diagnostic biomarkers and therapeutic targets for OA. Currently, a growing body of proteomic studies has been done to unveil a number of inflammatory cytokines, proteases, and cartilaginous matrix cleavages in the blood serum, synovial fluid, and articular cartilage from OA patients. Little information is available regarding the protein profiles of disturbances at subchondral bone in the pathophysiology of OA. The technical difficulties in protein extraction from tissues particularly bone and quantitative analyses of protein profile are discussed; cellular proteomics of the defective osteoblasts and secretomics for the osteoblasts-chondrocytes crosstalk are proposed to supplement the information obtained from the bone tissue proteomics.

Diffusion tensor imaging of somatosensory tract in cervical spondylotic myelopathy and its link with electrophysiological evaluation.

BACKGROUND AND CONTEXT: Abnormal somatosensory evoked potential (SEP) (ie, prolonged latency) has been associated with poor surgical prognosis of cervical spondylotic myelopathy (CSM). PURPOSE: To further characterize the extent of microstructural damage to the somatosensory tract in CSM patients using diffusion tensor imaging (DTI). STUDY DESIGN/SETTING: Retrospective study. PATIENT SAMPLE: A total of 40 volunteers (25 healthy subjects and 15 CSM patients). OUTCOME MEASURES: Clinical, electrophysiological, and radiological evaluations were performed using the modified Japanese Orthopedic Association (mJOA) scoring system, SEP, and cord compression ratio in anatomic magnetic resonance (MR) images, respectively. Axial diffusion MR images were taken using a pulsed gradient, spin-echo-echo-planar imaging sequence with a 3-T MR system. The diffusion indices in different regions of the spinal cord were measured. METHODS: Comparison of diffusion indices among healthy and myelopathic spinal cord with intact and impaired SEP responses were performed using one-way analysis of variance. RESULTS: In healthy subjects, fractional anisotropy (FA) values were higher in the dorsal (0.73±0.11) and lateral columns (0.72±0.13) than in the ventral column of white matter (0.58±0.10) (eg, at C4/5) (p<.05). FA was dramatically dropped in the dorsal (0.54±0.16) and lateral columns (0.51±0.13) with little change in the ventral column (0.48±0.15) at the compressive lesions in CSM patients. There were no significant differences in the mJOA scores or cord compression ratios between CSM patients with or without abnormal SEP. However, patients with abnormal SEP showed an FA decrease in the dorsal column cephalic to the lesion (0.56±0.06) (ie, at C1/2, compared with healthy subjects [0.66±0.02]), but the same decrease was not observed for those without a SEP abnormality (0.67±0.02). CONCLUSION: Spinal tracts were not uniformly affected in the myelopathic cervical cord. Changes in diffusion indices could delineate focal or extensive myelopathic lesions in CSM, which could account for abnormal SEP. DTI analysis of spinal tracts might provide additional information not available from conventional diagnostic tools for prognosis of CSM.

Quantitative analysis of fiber tractography in cervical spondylotic myelopathy.

BACKGROUND CONTEXT: Diffusion tensor fiber tractography is an emerging tool for the visualization of spinal cord microstructure. However, there are few quantitative analyses of the damage in the nerve fiber tracts of the myelopathic spinal cord. PURPOSE: The aim of this study was to develop a quantitative approach for fiber tractography analysis in cervical spondylotic myelopathy (CSM). STUDY DESIGN/SETTING: Prospective study on a series of patients. MATERIALS AND METHODS: A total of 22 volunteers were recruited with informed consent, including 15 healthy subjects and 7 CSM patients. The clinical severity of CSM was evaluated using modified Japanese Orthopedic Association (JOA) score. The microstructure of myelopathic cervical cord was analyzed using diffusion tensor imaging. Diffusion tensor imaging was performed with a 3.0-T magnetic resonance imaging scanner using pulsed gradient, spin-echo, echo-planar imaging sequence. Fiber tractography was generated via TrackVis with fractional anisotropy threshold set at 0.2 and angle threshold at 40°. Region of interest (ROI) was defined to cover C4 level only or the whole-length cervical spinal cord from C1 to C7 for analysis. The length and density of tracked nerve bundles were measured for comparison between healthy subjects and CSM patients. RESULTS: The length of tracked nerve bundles significantly shortened in CSM patients compared with healthy subjects (healthy: 6.85-77.90 mm, CSM: 0.68-62.53 mm). The density of the tracked nerve bundles was also lower in CSM patients (healthy: 086±0.03, CSM: 0.80±0.06, p<.05). Although the definition of ROI covering C4 only or whole cervical cord appeared not to affect the trend of the disparity between healthy and myelopathic cervical cords, the density of the tracked nerve bundle through whole myelopathic cords was in an association with the modified JOA score in CSM cases (r=0.949, p=.015), yet not found with ROI at C4 only (r=0.316, p=.684). CONCLUSIONS: The quantitative analysis of fiber tractography is a reliable approach to detect cervical spondylotic myelopathic lesions compared with healthy spinal cords. It could be employed to delineate the severity of CSM.

HIF-1α/VEGF signaling pathway may play a dual role in secondary pathogenesis of cervical myelopathy.

Cervical spondylotic myelopathy (CSM) is one of the most common spinal cord disorders affecting the elderly. Yet the exact pathophysiology of CSM remains unclear. Vascular response to initial mechanical compression and associated ischemia may involve in secondary pathophysiology. Chronic compressive lesions to cervical cord resulting in lack of perfusion have established considerable evidences to support ischemia as an important pathogenesis both in patients and animal models, a similarity as that of acute spinal cord injury (SCI). In hypoxic condition following SCI, the up-regulation of vascular endothelial growth factor (VEGF), is consistent with increasing hypoxia induced factor-1α (HIF-1α) in acute periods. HIF-1α/VEGF signaling pathway is thought to play a dual role following SCI. In one hand, VEGF was demonstrated to be correlated with angiogenesis (protecting vascular endothelial cells, increasing blood vessel density and improving regional blood flow), neurogenesis (antiapoptotic, neurotrophic, attenuate axonal degradation), and locomotor ability improvement. In other hand, some studies revealed that VEGF have limited therapeutic effect, even exacerbate the secondary damage following SCI. VEGF administrations in acute or subacute periods result in elevation of blood-spinal cord barrier (BSCB) permeability even last for chronic course. BSCB permeability elevation initiates a secondary cascade of events involving excitotoxicity, infiltration of leukocytes and tissue edema. With comprehensive understanding of temporal and spatial of HIF-1α/VEGF signaling pathway, development of therapeutic strategies to promote new vessel growth while minimize the deleterious effects of VEGF-induced microvascular permeability, and thereby improve neurologic function, seems to be feasible and promising.

Somatosensory-evoked potentials as an indicator for the extent of ultrastructural damage of the spinal cord after chronic compressive injuries in a rat model.

OBJECTIVE: Somatosensory-evoked potentials (SEPs) were found to correlate well with the disability and postoperative recovery in patients with cervical spondylotic myelopathy. Yet the exact pathophysiology behind it remains to be elucidated. This study aims to characterise the ultrastructural changes of a chronically compressive spinal cord with various SEP responses in a rat model. METHODS: A total of 15 rats were used with surgical implantation of a water-absorbing polymer sheet into the cervical spinal canal on the postero-lateral side, which expanded over time to induce chronic compression in the cord. At postoperative 6 months, the functional integrity of the cords was recorded by SEP responses by comparing injured and non-injured sides, and the ultrastructural integrity was assessed by 7-T magnetic resonance (MR) diffusion imaging, contrast-enhanced micro-computed tomography (µCT) and histological evaluations. RESULTS: Six rats showed unchanged SEP, and the other nine showed decreased amplitude only (n=5) or delayed latency (n=4). The circulation insults of the cords were found among all the rats, showing central canal enlargement, intra-tissue bleeding or increased blood vessels in the central grey matter. Ultrastructural damage was noted in the rats with changed SEP responses, which was suggested by lower fractional anisotropy and higher contrast intensity radiologically and echoed by less myelin stain and cavitation changes histologically. In the animals with delayed latency, the cord showed significant loss of motoneurons as well as gross appearance distortion. CONCLUSIONS: The categorised SEP responses by amplitude and latency could be an indicator for the extent of ultrastructural damage of the spinal cord after chronic compressive injuries. SIGNIFICANCE: The findings built a solid foundation for SEP application in clinical diagnosis and prognostication of spinal cord injuries.

The use of brushite calcium phosphate cement for enhancement of bone-tendon integration in an anterior cruciate ligament reconstruction rabbit model.

This study was designed to investigate the osteoconductivity and bioresorption of brushite calcium phosphate cement (CPC) in bone-tendon interface healing after anterior cruciate ligament (ACL) reconstruction. Surgical reconstruction using grafted tendon in bone tunnel was performed bilaterally in 28 skeletal mature rabbits. Brushite CPC was implanted between grafted tendon and bone tunnel of one limb with the contralateral one as the control. A batch of 14 rabbits was sacrificed at 6 and 12 weeks, respectively, after surgery. At each time point, six rabbits were used for micro-CT and subsequent histological examinations, whereas the remaining eight rabbits were used for pull-out testing. The components of brushite CPC-dicalcium phosphate dihydrate matrix degraded rapidly with beta-tricalcium phosphate granules left for guiding new bone formation. Brushite CPC augmented the peri-tendon bone volume and promoted bone growth into the healing interface. The ultimate strength and stiffness of the graft-tunnel complexes on experimental side was higher than that of the control by 117% and 102%, respectively, at 6 weeks postoperatively (p < 0.05 for both). The use of brushite CPC caused a paradigm shift in failure mode from intra-tunnel to intra-articular portion at 12 weeks postoperatively (p = 0.013). Brushite CPC significantly enhanced the bone-tendon integration after ACL reconstruction, which provided a scientific basis for clinical application.

Peri-graft bone mass and connectivity as predictors for the strength of tendon-to-bone attachment after anterior cruciate ligament reconstruction.

The present study was designed to compare peri-graft bone mass and microarchitecture with tendon-to-bone (T-B) attachment strength after anterior cruciate ligament (ACL) reconstruction in a rabbit model. Surgical reconstruction using digital extensor tendon in bone tunnel was performed on 58 rabbits. Forty-two of the 58 rabbits were sacrificed at week 0, 2, 6 and 12 after operation respectively. The femur-graft-tibia complexes were harvested for pQCT and micro-CT examination to characterize the spatiotemporal changes of peri-graft bone in T-B healing in conjunction with histological examination. The remaining 16 rabbits were euthanized at week 6 and 12 postoperatively (i.e. 8 rabbits for each time point) for pull-out test after micro-CT examination to investigate the relationship between the T-B attachment strength and peri-graft bone mass/microarchitecture. Peri-graft BMD, BV/TV and connectivity was significantly lower at week 6 than those at time zero although there were no significant changes detected in the first 2 postoperative weeks. In addition, peri-graft bone mass and connectivity was significantly lower on the tibial side than those on the femoral side; and osteoclasts accumulated on the surface of peri-graft bone. Grafted tendon was prone to be pulled out from the tibial tunnel with bone attachment; the weakest point of the complexes shifted from the healing interface at time zero to peri-graft bone at week 6 after operation. With reverse of peri-graft bone at week 12 postoperatively, the weakest point shifted to the intra-osseous tendinous portion. The stiffness of T-B attachment correlated with peri-graft BV/TV (r2 = 0.68, p = 0.001) and connectivity (r2 = 0.47, p = 0.013) at week 6 after operation. T-B healing was a highly dynamic process of emergence and maintenance of peri-graft bone. T-B attachment strength was in relation to peri-graft bone mass and connectivity after ACL reconstruction. The measurement of peri-graft bone should be useful to monitor the quality of T-B healing and guide the postoperative rehabilitation.

Influence of bone adaptation on tendon-to-bone healing in bone tunnel after anterior cruciate ligament reconstruction in a rabbit model.

Anterior cruciate ligament (ACL) reconstruction with placement of grafted tendon in bone tunnel is a common surgical procedure. Bone tunnel creation may result in stress shielding of postero-lateral regions of tibial tunnel. The present study was designed to characterize the changes of peri-graft bone and compare with tendon-to-bone (T-B) healing in spatial and temporal manners after ACL reconstruction in rabbit. Surgical reconstruction using digital extensor tendon in bone tunnel was performed on 48 rabbits. Twelve rabbits were sacrificed at 0, 2, 6, and 12 weeks postoperatively for radiological and histological examinations. Bone mass and microarchitecture at the anterior, posterior, medial, and lateral regions of tunnel wall at distal femur and proximal tibia were evaluated. Using peripheral quantitative computed tomography, a 26, 22, and 42% decrease in bone mineral density (BMD) relative to baseline was present in the medial region of the femoral tunnel and the posterior and lateral regions of the tibial tunnel, respectively, at week 12 postoperatively (p < 0.05). It was accompanied by a decrease in trabecular number and increase in trabecular spacing, the shift of platelike to rodlike trabeculae, and loss of anisotropy under micro-computed tomography evaluation. This finding was echoed by histology showing increased osteoclastic activities and poor T-B healing in these regions. In conclusion, the postoperative bone loss and associated poor T-B healing was region-dependent, which may result from adaptive changes after tunnel creation.